WO2016115376A1 - Detection and treatment of double drug resistant melanomas - Google Patents

Abstract

Methods of predicting or detecting the development of acquired resistance to therapeutic effects of combined B-RAF/MEK inhibitor therapy in a patient suffering from cancer are provided. The method comprises assaying a sample obtained from the patient for a measure of combined B-RAF/MEK inhibitor therapy resistance, selecting samples that exhibit a measure of resistance identified in the assaying step; and identifying a patient whose sample was selected in the selecting step as susceptible to developing resistance to combined B-RAF/MEK inhibitor therapy. Treatment strategies can be based on predicting or detecting the development of acquired resistance to therapeutic effects of combined B-RAF/MEK inhibitor therapy in a patient suffering from cancer.

Description

DETECTION AND TREATMENT OF DOUBLE DRUG RESISTANT MELANOMAS

[001] This application claims the benefit of U.S. provisional patent application number 62/103,517, filed January 14, 2015, the entire contents of which are incorporated herein by reference. Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[002] This invention was made with Government support of Grant No. CA176111 , awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

[003] The present invention relates generally to detection, diagnosis, monitoring and treatment of cancer, such as melanoma. The invention more specifically pertains to cancers resistant to combination therapy using inhibitors of B-RAF and MEK, and selection of effective treatment strategies.

BACKGROUND

[004] RAS and BRAF are frequently mutated in human malignancies. In advanced melanoma, NRAS and, less often, KRAS mutations occur in about 20% of cases and are mutually exclusively with BRAF mutations, which are present in about 50% of cases. Somatic MEK1 or MEK2 mutations, which can be concurrent with RAS or BRAF mutations, have also been detected (Hodis et al., 2012; Krauthammer et al., 2012; Nikolaev et al., 2012; Shi et al., 2012a), but their roles in pathogenesis and therapeutic responses remain ill defined. BRAF mutations strongly predict responses to ATP-competitive BRAF inhibitors (BRAFi) such as vemurafenib and dabrafenib. Allosteric MEK1 and MEK2 inhibitors (MEKi), such as trametinib, selumetinib, cobimetinib, and binimetinib, may have antitumor activities against a broader melanoma segment, including those with NRAS mutations or with both wild-type (WT) NRAS and WT BRAF, but MEKi monotherapy for patients with BRAF mutant

melanomas is associated with a narrower therapeutic window (versus BRAFi) (Ribas and Flaherty, 2011).

[005] Melanoma regrowth after initial response to MEKi has been attributed to a P124LMEK1 mutation (Emery et al., 2009), and acquired MEKi resistance in BRAF mutant colorectal cell lines has been linked to a ^F129LMEK1 mutation (Wang et al., 2011) or BRAF amplification (Corcoran et al., 2010). How these MEK mutations mechanistically account for MEKi resistance is not entirely clear. Due to the superior clinical benefits of BRAFi for melanoma patients, mechanisms of acquired BRAFi resistance have been studied extensively, and those well validated clinically include NRAS or KRAS mutations (Nazarian et al., 2010; Shi et al., 2014), ^V600EBRAF amplification (Shi et al., 2012b) or alternative splicing (Poulikakos et al., 2011 ; Shi et al., 2012a), MEK1 or MEK2 mutations (Shi et al., 2012a; Wagle et al., 2011), CDKN2A loss (Shi et al., 2014), and genetic alterations in the phosphatidylinositol 3-kinase- phosphatase and tensin homolog-protein kinase B (PI3K-PTEN-AKT) pathway (Shi et al., 2014; Van Allen et al., 2014). The convergence of multiple mechanisms to reactivate the mitogen-activated protein kinase (MAPK) pathway provided a strong rationale for combined BRAF and MEK targeting to overcome BRAFi resistance, a strategy that is supplanting single-agent BRAFi therapy. However, acquired resistance to BRAFi+MEKi still limits the long-term survival of patients with advanced ^V600E/KBRAF melanoma. A priori, the

intransigence of acquired resistance in response to dual MAPK targeting may be due to preferential emergence of MAPK-redundant resistance pathways. Evidence of branched evolution, extensive interpatient and tumor heterogeneity, and increased tumor fitness as melanoma emerges from BRAFi-imposed evolutionary selection may help explain why the BRAFi+MEKi combinatorial approach is also an "uphill battle" (Shi et al., 2014).

[006] There remains a need for improved tools to permit the detection, identification and prognosis of drug resistant cancers, particularly B-RAF/MEK inhibitor-resistant melanomas. There also remains a need for targets useful in the detection and treatment of cancer.

SUMMARY

[007] The invention provides a method of predicting or detecting the development of acquired resistance to therapeutic effects of combined B-RAF/MEK inhibitor therapy in a patient suffering from cancer. In one embodiment, the method comprises assaying a sample obtained from the patient for a measure of combined B-RAF/MEK inhibitor therapy resistance, selecting samples that exhibit a measure of resistance identified in the assaying step; and identifying a patient whose sample was selected in the selecting step as susceptible to developing resistance to combined B-RAF/MEK inhibitor therapy. Typically, the measure of resistance is selected from:

(1) ^V600E _BRAF ultra-amplification;

(2) LOF ^F127VPTEN expression;

(3) homozygous PTEN deletions;

(4) ^G12RNRAS with selective mutant allele amplification;

(5) a homozygous CDKN2A deletion shown in Table 2;

(6) concurrent heterozygous ^Q61KNRAS with homozygous CDKN2A deletion;

(7) ^V600EBRAF amplification concurrent with homozygous CDKN2A deletion;

(8) ^V600EBRAF amplification concurrent with DUSP4 deletion; (9) ^V600EBRAF amplification concurrent with one or more MEK1 and MEK2 mutations selected from F53Y, Q56P/Q60P, K57N, K59del, V60E, I111S, C121S/C125S, P124IJS G128V, F129L, V154I, E203K, and G276W;

(10) homozygous CDKN2A deletion concurrent with homozygous PTEN deletion; (11) hemizygous PIK3R1 deletion; and

(12) a genetic profile listed in Table 2 as associated with double drug disease

progression (DD-DP);

[008] Examples of the assaying of the method include, but are not limited to, targeted sequencing, real-time RT-PCR, Sanger sequencing and/or whole exome sequencing. In one embodiment, the ^V600EBRAF ultra-amplification is identified by detecting more than 50 copies, or more than 60 copies, or more than 70 copies of ^V600EBRAF. In one embodiment, ultra- amplification of ^V600EBRAF is identified by detecting about 74 copies of ^V600EBRAF. The assaying can comprise, for example, contacting the sample with one or more primers selected from SEQ ID NOs: 1-106.

[009] In one embodiment, the combined B-RAF/MEK inhibitor therapy comprises treatment with a B-RAF inhibitor selected from vemurafenib and dabrafenib. In another embodiment, the combined B-RAF/MEK inhibitor therapy comprises treatment with a MEK inhibitor selected from trametinib, selumetinib, cobimetinib, and binimetinib.

[0010] In one embodiment, the sample is selected from tissue, bodily fluid, blood, tumor biopsy, spinal fluid, and needle aspirate. In one embodiment, the method is performed prior to treatment with combined B-RAF/MEK inhibitor therapy. In an another embodiment, the method is performed after treatment with combined B-RAF/MEK inhibitor therapy. In some embodiments, the method is performed during disease progression or clinical relapse on combined B-RAF/MEK inhibitor therapy. In yet another embodiment, the method is performed after suspension of combined B-RAF/MEK inhibitor therapy.

[0011] The cancer can be any cancer that develops resistance to combined BRAF/MEK inhibitor therapy. In one embodiment, the cancer is melanoma. Other cancers include, but are not limited to, lung cancer and other BRAF mutant cancers that are responsive to inhibitors of BRAF, MEK or ERK or their combinations.

[0012] In one embodiment, the method further comprises identifying in a patient who is susceptible to developing or has developed resistance to combined B-RAF/MEK inhibitor therapy the form(s) of resistance and method(s) to counter such resistance. Such methods to counter resistance driven by described mechanisms include (but are not limited to):

combinations based on BRAF and MEK inhibitors with the additions of: Inhibitors of the PI3K- AKT-mTORC pathway; Inhibitors downstream of the MAPK pathway such as CDK4/6 inhibitors; Inhibitors that block MEK activation by RAF; Inhibitors that block BRAF and MEK interactions; Inhibitors that block RAF dimerization; and Inhibitors that block RAS functionsln one embodiment, the method further comprises treating the patient with intermittent dosing of combined B-RAF and MEK inhibitors.

[0013] The invention additionally provides a method of preventing or suppressing acquired resistance to combined B-RAF/MEK inhibitor therapy in a patient. In one embodiment, the method comprises administering to the patient a therapeutic agent selected from the group consisting of:

(a) a suppressor of C-RAF kinase activity;

(b) an inhibitor of activation of MEK1 or MEK2 by C-RAF; and

(c) an inhibitor of activation of ERK1 or ERK2 by MEK1 and MEK2.

[0014] In one embodiment, the therapeutic agent is selected from the group consisting of: Omni-RAF, CRAF and RAF paradox breakers, and ERK inhibitors. In another embodiment, the therapeutic agent is CCT196969, CCT241161, PLX3397, PLX7904 and/or SCH442984.

BRIEF DESCRIPTION OF THE FIGURES

[0015] Figures 1A-1E. Melanomas Resistant to BRAF/MEK Inhibitors Harbor Exaggerated Genetic Mechanisms of BRAF Inhibitor Resistance. (1A) Clinical photos of patients denoting specific genetic mechanisms of drug resistance detected within specific tumors (see text). For patient #9, BRAFi-disease progressive melanomas responded to BRAFi+MEKi (arrows, examples) on day 14 with disease progression ensuing as evident on day 88. DP, disease progression on BRAFi; DD-DP, double drug-disease progression. (1 B) DD-DP melanoma from patient #2 harboring BRAF ultra-amplification by genomic DNA (gDNA) quantitative PCR (Q-PCR; averages of duplicates; two independent primer sets; PMN, peripheral mononuclear cells) and confirmed by Sanger sequencing (baseline tumor, heterozygous V600E allele). (1C) DD-DP melanoma from patient #6 with concurrent heterozygous Q61KNRAS,

K197*PTEN non-sense allele (not expressed), and F271VPTEN loss-of-function allele (expressed, RNASeq data shown). Display by Integrative Genome Viewer with Sanger validatation. (1D) G12RNRAS homozygosity in the DD-DP tumors from patient #9. (1E) G12RNRAS copy number gains in both DD-DP tumors from patient #9. (Note shades of gray in peaks correspond to indicated nucleotides.) Display by Circos. See also Figure 9.

[0016] Figures 2A-2K. Melanoma Cells with Acquired BRAFi Resistance Further Resist BRAFi+MEKi by Augmenting Existing or Combining Distinct Mechanisms. (2A) Relative drug exposure times required to achieve resistance to BRAFi+MEKi in three isogenic groups of cell lines comparing progression from SDR->DDR vs. P->DDR (P, parental, SDR, Single Drug or BRAFi Resistant; DDR, Double Drug Resistant). BRAFi, vemurafenib; MEKi, selumetinib; [inhibitor] range from 0.1 to 2.0 μΜ at indicated increments. (2B) gDNA and cDNA BRAF copy numbers as measured (average of duplicates) by Q-PCR (two sets of primers) or Q-RT-PCR (two reference genes) (confirmed by semi-quantitative PCR; see agarose gel). PMN, peripheral mononuclear cells. (2C) gDNA and cDNA NRAS levels in the M249 isogenic triplet in A. (2D) Sanger sequencing of cDNAs with chromatograms (note shades of gray in peaks correspond to indicated nucleotides) showing detection of the WT vs. mutant NRAS transcripts (ratio estimated by peak heights). (2E) Western blot of indicated total and phospho-protein levels from three isogenic triplets (SDR sub-lines annotated with known BRAFi resistance mechanisms; FL, full-length, TR, truncated; TUBULIN, loading control). SDR and DDR sub-lines have been treated respectively with BRAFi or BRAFi+MEKi (1 μΜ) 16h prior to lysate preparation. Quantification of WB signals for NRAS (M249 triplet): 1 , 0.98, 1.65, for p61 BRAF (M397 triplet): 1 , 2.55, 7.33, and for FL BRAF/p-CRAF (M395 triplet): 1, 10.89, 13.63 and 1 , 9.89, 11.89 (densitometry signals of targets normalized to those of TUBULIN; all values are normalized to the parental values). (Figs. 2F-2H) MUTNRAS up- expression confers DDR. (2F) NRAS knockdown by shRNA as shown by Western blotting 72h after lentiviral infections. M249 SDR and DDR were treated with their respective inhibitor(s) at 1 μΜ. shSCR, shScrambled. (2G) Three-day MTT assays (error bars, SEM, n=5; normalized to DMSO as 100%). BRAFi (vemurafenib), MEKi (selumetnib) in μΜ. (2H) Ten-day clonogenic drug survival assays. BRAFi or BRAFi+MEKi treatments every two days were started 24h after plating. (Figs. 2I-2K) Combination of Q61KNRAS and PTEN loss magnifies DDR. (2I) The M238 AR, Acquired Resistance (to BRAFi). cDNA Sanger sequencing with chromatogram showing WT vs. mutant NRAS transcripts (ratio estimated). (2J) Stable knockdown of PTEN by lentiviral shRNA in M238 AR (SDR) showing the levels of indicated phospho- and total proteins by Western blotting of cellular lysates 72h

posttransduction. BRAFi, 1 μΜ; GAPDH, loading control. (2K) Three-day MTT assays as in (2G).

[0017] Figures 3A-3H. Melanoma Cells Clonally Develop Resistance to Upfront BRAFi+MEKi via Alternative Genetic Configurations. (3A) Three-day MTT assays (error bars, SEM, n=5; top, relative raw values; bottom, normalized to DMSO vehicle as 100%). The M249 triplet cell lines were plated 16h without inhibitors (BRAFi-vemurafenib; MEKi-selumetinib; ERKi-

SCH772984; in μΜ). (3B) Western blots (WBs) of indicated total and phospho-proteins. Cells were plated 16h without inhibitors prior to treatments for 1h (0-10 μΜ in 10-fold increments). TUBULIN, loading control. (3C) BRAF copy number by gDNA Q-PCR (averages, duplicates; two primer sets). (3D) Sanger sequencing chromatograms showing BRAF and MEK1 mutational status and their allelic ratios (note shades of gray in peaks correspond to indicated nucleotides). (3E) Integrated Genome View (IGV) snapshots of reference and mutant/variant allelic frequencies (MAF) centered on the A to T mutation (chromsome 7:140453136; V6∞E_BRAF) and on the C to G mutation (chromosome 15:66729179; ^F129LMEK1). MutantWT estimated from the MAFs. Note a low MAF of ^F129LMEK1 in M249 P. (3F) Distinct BRAF amplicons in DDR4 vs. 5 (top). CNVs, displayed by Circos (with respect to M249 P). MEK1 copy number gain in only DDR5. (3G) WBs of indicated total and phospho-proteins from cells plated 16h without inhibitors prior to ERKi treatments for 1h (0, 0.01, 0.1, 1.0, and 10 μΜ) without (left) or with (right) BRAFi+MEKi (1 μΜ). (3H) The M249 isogenic triplet were plated 16h without inhibitors. Only DDR4/5 were treated (1h) with BRAFi+MEKi (1 μΜ) prior to the lysates being analyzed by WBs. See also Figure 10.

[0018] Figures 4A-4I. Achieving BRAF/MEK Inhibitor Resistance via Tuning ^V600EBRAF Gene Dosage with or without MEK Mutations. (4A) M249 DDR4/5 (plated 16h with BRAFi+MEKi, 1 μΜ; transduced with lentiviral shVector or shBRAF for 48h; and treated with the indicated inhibitors at 1 μΜ for 1h) were analyzed by Western blots (WBs) (TUBULIN, loading control). (4B) WBs of the M249 triplet -/+ CRAF or BRAF knockdown (48h post lentiviral transduction). (4C) Cells from B were plated for clonogenic assays. (4D) Whole exome-based phylogenetic relationships of the M249 triplet cell lines. Branch lengths, proportional to the number of heterozygous single nucleotide variants and small insertion-deletions private to each cell line with respect to the theoretical common ancestral cell sub-population (designated #1). The DDRunique copy number variations of indicated genes also shown. (Figs. 4E, 4F) WBs of total and phospho-protein levels in M249 triplet and M249 P engineered to express ^V600EBRAF and ^F129LMEK1 (4E) or ^Q56PMEK1 (4F). Selected cell lines as indicated were treated with BRAFi+MEKi (1 μΜ) for 16h and then washed free of inhibitors for 8h. (4G) M249 P engineered to express vector, ^WTBRAF, or V6OOE/R509^HBRAF (without inhibitors) or M249 DDR4 (BRAFi+MEKi, 1 μΜ, 16h) were analyzed by WBs. (4H) Clonogenic assays of M249 P engineered to express the indicated levels of WT vs. mutant BRAF and/or MEK1. Relative resistance to BRAFi+MEKi were measured over the indicated concentration range and over durations. (4I) Temporal genetic clonal evolution of MAPKi resistance with magnitudes matching graded selective pressures. Single MAPKi, BRAFi; double MAPKi, 47 BRAFi+MEKi; circle, dominant tumor or cell line sub-clone. Distinct ^V600EBRAF amplicons, convergent evolution. Augmented gene dosage or combinatorial genetic alterations are proposed as alternative clonal evolutionary pathways to acquire resistance. See also Figures 11 and 12.

[0019] Figures 5A-5F. Distinct MEK Mutants Share Enhanced Interaction with ^V600EBRAF (5A, 5B, 5C) The M249 triplet cell lines were plated without (P) or with (DDR4/5)

BRAFi+MEKi (1 μΜ, 16h), and lysates were subjected to immunoprecipitation (IP) using the isotype or BRAF- (5A), MEK1- (5B), and MEK2- (5C) specific antibodies. The

immunoprecipiated and total fractions probed by Western blots (WBs). TUBULIN, loading control. (5D) WB analysis of total and phospho-MEK1/2 and -ERK levels. (5E) Structure of MEK1 (two views, 180° rotated) indicating the locations (lighter gray) of mutations in MEK1 (or residues homologous to mutations in MEK2). All mutations, except I111S and P124S, have been detected specifically in melanomas with clinical MAPKi acquired resistance. (5F) M249 P engineered to express vector or FLAG-^WTMEKI, -^F129LMEK1, - ^Q56PMEK1 concurrent with over-expression of either HA-^WTBRAF or HA^V600EBRAF were plated with BRAFi+MEKi (1 μΜ, 16h; except vector control), and the lysates were subjected to IP (anti-lgG or -FLAG). WBs of IP and total fractions. See also Figure 13.

[0020] Figures 6A-6E. A BRAF-MEK Interface Critical for ^V600EBRAF-^MUTMEK1 Interaction and Cooperativity in Conferring Double Drug Resistance. (Figs. 6A-6B) BRAF arginine 662 at a MEK1-BRAF interface. (6A) A predicted MEK1- ^V600EBRAF complex (KD, kinase domain) highlighting the locations of (i) MEK1 amino acid residues mutated in melanomas with acquired MAPKi resistance, (ii) ^V600EBRAF R509, critical for RAF-RAF dimerization, (iii) v^BRAF R662, structurally homologous to KSR2 A879 critical for MEK1-KSR2 interaction, and (iv) ^V600EBRAF 1617, critical for MEK-BRAF dimerization. (6B) Zoomed-in details of a MEKI-^V600EBRAF interfaces, highlighting MEK1 activation segment residues (M219, S222, and V224) interacting with ^V600EBRAF R662, 1617 and I666, and interactions predicted to be abolished by a R662L mutation. (6C) Transfection of indicated HA-tagged BRAF constructs (vs. vector) in human 293T cells and WBs of lysates. TUBULIN, loading control. (6D) M249 P engineered to moderately over-express HA-BRAF or the indicated mutant versions along with either FLAG-MEK1 mutant (F129L or Q56P). Experiments were performed as described for Figure 5F. (6E) Clonogenic assays of M249 P engineered to express WT vs. indicated mutant BRAF, resistance-associated MEK1 mutants, and/or their empty vectors (Vec).

Relative resistance to BRAFi+MEKi assessed over the indicated concentration range and time points. Three repeats (for 0.1 and 1.0 μΜ) are shown, and growths were quantified (1 μΜ; n=3; normalization relative to ^V600EBRAF+^MUTM EK1 transduced cells as 100%; means and SD of the mean; *p < 0.05, ***p < 0.001 , ns, not significant based on ANOVA). See also Figure 14.

[0021] Figures 7A-7F. Resistance to Combined BRAF/MEK Inhibition Results in Exquisite Drug Addiction (7A) Clonogenic survival and degrees of drug addiction. Cell were plated (BRAFi+MEKi, 1 μΜ; 48 h) and cultured (9 d) with or without specific inhibitor withdrawal (data shown representative of three independent repeats). (Figs. 7B, 7C) Clonogenic/drug addiction assays as in A except for the indicated high (7B) or low (7C) ERKi (SCH772984) doses starting at 48h. (7D) Clonogenic/drug addiction assays comparing SDR vs. DDR cell lines of distinct genetic backgrounds and resistance mechanisms. (7E) Western blot analysis of baseline and rebound p-ERK levels in response to acute BRAFi (SDR) or BRAFi+MEKi (DDR) withdrawal for 4 and 24h. TUBULIN, loading control. Quantification of p-ERK signals normalized to TUBULIN levels is shown for each cell line relative to the baseline. (7F) Strong positive correlation between rebound p-ERK levels upon inhibitor(s) withdrawal (0 vs. 4 h) (7E) and the degree of drug addiction (quantification of differential clonogenic growths in 7D). See also Figures 15 and 16.

[0022] Figure 8. ^V600EBRAF-CRAF-MEK Signalsome Upregulation with Opposite Impacts on Melanoma Fitness Contingent on BRAF/MEK Inhibitor Presence. ^V600EBRAF melanoma cells can upregulate a ^V600EBRAF-CRAF-MEK signalsome in response to selection by

BRAFi+MEKi. This resistansome can consist of a supra-physiologic level of ^V600EBRAF, which spuriously activate CRAF. Alternatively, moderately over-expressed ^V600EBRAF concomitant with a mutant MEK1/2 can lead to increased ^V600EBRAF-^MUTMEK interaction. Both signaling configurations strongly favor ERK activation, leading to growth/survival finely tuned to the BRAFi+MEKi level. Paradoxically, acute removal of BRAFi+MEKi disrupts this fine tuning and results in a p-ERK rebound favoring cell arrest/death (i.e., drug addiction). WT (grey rectangles) and mutant (dark rectangles) proteins; BRAFi or MEKi, dark circles.

[0023] Figures 9A-9B. Related to Figure 1. The novel F271VPTEN mutation results in loss- of-function. (9A) Structure of PTEN (WT, lighter; F271V mutant, darker) showing locations of V271 (thick black arrow) and F271 (thick white arrow) as well as those of amino acids within 4A (three thin arrows). (9B) Western blot showing that PTEN WT expression in the PTEN non-expressing M249 BRAF mutant melanoma cell line resulted in p-AKT suppression whereas PTEN F271V expression did not. TUBULIN, loading control.

[0024] Figures 10A-10H. Related to Figure 3. (10A) Spontaneous recovery of ERK signaling in the M249 triplet inhibitor-sensitive and -resistant cell lines in the continuous presence of BRAFi and MEKi. Cells were plated in the absence of both inhibitors (BRAFi, 1 μΜ

vemurafenib; MEKi, 1 μΜ selumetinib) for 16 hours, and, subsequently, were treated with vehicle (DMSO) or both inhibitors. Cellular lysates were collected at the indicated time points after inhibitor treatment and analyzed for the indicated phospho- and total protein levels by Western blotting. TUBULIN, loading control. (10B) Activation-associated phosphorylation levels of RSK and ERK are dynamically and positively correlated in M249 double drug- resistant sub-lines. Western blot analysis of lysates from M249 isogenic lines for indicated total and phospho-proteins (p-p90RSK Thr573). Cells were plated 16h without inhibitors followed by BRAFi+MEKi (1 μΜ) treatments for the indicated durations (0, 1, 6, and 24 hours). (Figs. 10C-10E) M249 sub-lines derived to resist a fixed and high BRAFi+MEKi concentration also harbor concomitant BRAF and MEK1 genetic alterations. (10C) M249 parental drug-naive cells were treated from the outset with BRAFi (vemurafenib 0.5 μΜ) and MEKi (selumetinib 0.5 μΜ) every 2-3 days for over three month, and two independent double drug-resistant proliferative subpopulations were isolated and designated as DDR2 and DDR3. Genomic DNA (gDNA) were isolated from M249 parental and DDR2/3 and analyzed for

BRAF copy number with respect to normal diploid cells by quantitative PCR (Q-PCR) (values represent averages of duplicates as measured by two sets primers). (10D) Sanger gDNA sequencing chromatograms showing MEK1 mutational status and their approximate allelic ratios in the M249 isogenic double drug resistant (DDR) sub-lines (note shades of gray in peaks correspond to indicated nucleotides). Increased mutant (mut) relative to wild type (wt) MEK1 detection suggested selective copy number gain of MEK1 gDNA. (10E) MEK1/2 gDNA QPCR showing MEK1 vs. MEK2 copy numbers in M249 DDR sub-lines relative to the parental M249. M249 DDR4, MEK1 wt; M249 DDR5, MEK1 F129L. Values represent averages of four measurements performed in two independent experiments; error bars, standard deviation. MEK1 p-values, 0.006/0.002 (primer sets 1/2, DDR2 vs. P); 0.09/0.002 (DDR3 vs. P); 0.29/0.59 (DDR4 vs. P); 0.04/0.003 (DDR5 vs. P). (Figs. 10F-10G) Cellular sensitivities of MAPKi resistance-associated MEK1 mutants to MEKi treatment in the absence of mutant BRAF. (10F) Indicated FLAG-tagged MEK1 constructs were introduced into HEK293T cells by transfection, and, 72 h after transfection, the cells were treated with MEKi/AZD6244/selumetinib for 1 hour at 0, 0.01, 0.1, 1.0 and 10 μΜ. Cellular lysates were analyzed by Western blotting for the indicated phospho- and total protein levels. Shown are different exposure snap shots (seconds) of p-ERK levels which were quantified by NIH Image J. (10G) Quantification of p-ERK levels normalized to TUBULIN levels in 10F. (10H) CRAF activation in double drug-resistant M249 sub-lines does not require continuous BRAF/MEK inhibitor treatment and ERK-mediated negative feedback. DDR4/5 were plated in the presence of both vemurafenib and selumetinib (both at 1 μΜ) for 20 hours followed by drug withdrawal for the indicated durations (hours).

[0025] Figures 11A-11F. Related to Figure 4. (11A) ^V600EBRAF over-expression in the double drug-resistant melanoma cell line M395 is required for CRAF activation. M395 DDR cells were infected with either shVECTOR or shBRAF lentivirus and cultured in the presence or absence of BRAFi (vemurafenib, 1 μΜ) and MEKi (selumetinib, 1 μΜ). Western blot analysis showed that BRAF knockdown in the absence of dual inhibitors did not diminish p-ERK levels while in the presence of dual inhibitors BRAF knockdown abrogated both p-CRAF and p-ERK levels. TUBULIN, loading control. (Figs. 11B-11 F) ^V600EBRAF gene dosage gain with or without a MEK1 mutation confers resistance to BRAF/MEK inhibitors but not double-drug addiction. (11 B) Western blot analysis of lysates from cell lines from A and B for indicated total and phospho-proteins levels showing the impact of inhibitor pre-conditioning on accelerating p-ERK rebound (parental cell p-ERK rebounds in days). Cells were plated for 24h without inhibitors, treated for 16h (except Parental or Parental+Vector) with BRAF and MEK inhibitors (1 μΜ) followed by inhibitor wash-out and continued incubation for 8h. (11C) Three-day drug exposure survival (MTT) assays (error bars, SEM, n=5; normalized to DMSO vehicle as 100%). The M249 isogenic triplet cell lines were plated 16h without inhibitors and then exposed to the indicated combinations of inhibitors (BRAFivemurafenib; MEKi- selumetinib). Experiment performed in parallel with that in B. (11D) Three-day drug exposure survival (MTT) assays for the M249 parental cell line engineered with the indicated constructs; the indicated expression of these constructs were initiated by a prior two day withdrawal from doxycycline. A parallel set of engineered cell lines were withdrawn from doxycycline and cultured or pre-conditioned in the presence of BRAF and MEK inhibitors (1 μΜ) for 28 days prior to the MTT experiment. (11 E) Scatter plot showing measurements (n=5) of the relative viability and growth of M249P engineered lines (as in 11 B) in the absence of BRAF/MEK inhibitors (- or + indicates prior pre-conditioning with combined BRAF and MEK inhibitors). All lines were removed from doxycycline, and data were expressed in each of three groups relative to an arbitrary value (from the non-preconditioned group) set as 100%. (11 F) Long-term (10 day clonogenic assay) and short-term (3 day MTT assay) growth capacities of indicated cell lines in the absence of both BRAF and MEK inhibitors. Raw MTT values (minus background, media only) at day 1 (24 h after seeding) and day 4 for each cell line are plotted as means (n=5) and their standard deviations. Numeric values stacking on top of day 4 values represent the fold changes in growth of each cell line (day 4 mean divided by day 1 mean).

[0026] Figures 12A-12C. Related to Figure 4. Both ^V600EBRAF over-expression and MEK1 mutation facilitate resistance to combined BRAF/MEK inhibitors. (12A) M395R, a BRAFi- resistant sub-line derived from M395 parental (P) by chronic selection with BRAFi and shown to resist BRAFi by ^V600EBRAF amplification and over-expression, was engineered by lentiviral transduction for the stable expression WT or mutant versions of FLAG-MEK1. Levels of indicated proteins and phospho-proteins were assessed by Western blotting using lysates from cells treated with BRAFi (vemurafenib) or vehicle (DMSO) for 48h hours (h). TUBULIN, loading control. (12B) M395 isogenic cell lines and M395R-engineered cell lines as in A were either treated with DMSO (P) or BRAFi+MEKi (vemurafenib+selumetinib) for 16h or washed free of inhibitors followed by additional 8h of culture. Western blot analysis of given proteins and dynamic p-ERK levels. (12C) Three-day MTT (top) and twelve-day clonogenic (bottom) drug survival assays, using BRAFi alone, MEKi alone, or both BRAFi+MEKi (error bars, SEM, n=5; top, normalized to DMSO vehicle as 100%). At 1 μΜ of BRAFi+MEKi, in both assays, M395R cell lines engineered to express either MEK1 mutant, survived significantly better than M395R expressing WT MEK1 or M395R (p < 0.001).

[0027] Figure 13. Related to Figure 5. Interaction between wild type and mutant MEK1 and BRAF. The M249 parental cell line was engineered to express vector or FLAG-MEK1 WT, C121S, E203K, K59del, 1111S concurrent with overexpression of either HA-^WTBRAF or HA- ^V600EBRAF. The stable cell lines were then plated with BRAFi+MEKi (1 μΜ) for 16h (except vector control) and lysed. The cellular lysates were subjected to immunoprecipitation using the isotype (IgG) or FLAG-specific antibodies. The immunoprecipiated (IP) and total fractions were then probed by Western blots as indicated. [0028] Figures 14A-14D. Related to Figure 6. A MEK1-^V600EBRAF complex with arginine 662 at an intermolecular interface. (14A) Structure of the ^WTMEKI-^WTBRAF heterodimer (KD, kinase domain) (PDB4MNE) showing (i) the face-to-face configuration of the complex, (ii) locations (amino acid residues highlighted with lighter gray near white arrow) of MEK1 residues mutated in melanomas with acquired BRAFi or BRAFi+MEKi resistance, and (iii) locations of BRAF V600 and R509 (i.e., the RAF dimerization interface; thin black arrows). MEK1 residues 1-65 were not resolved in the crystal structure of 4MNE. Hence, structure of this N-terminal MEK1 region was modeled by alignment to the MEK1 structure in PDB2Y4I. (14B) Structure of the ^WTMEKI-^V600EBRAF heterodimer modeled using the vemurafenib-bound ^V600EBRAF structure (PDB30G7) aligned with the ""^"BRAF shown in (14A) (root mean square distance or RMSD, 0.511 A). Circled, aC helix pulled up into a "closed" conformation; BRAF E600 residue, not resolved in PDB4MNE. (14C) A hydrophobic core critical for MEK1-KSR2 interaction (PDB2Y4I) showing the aG helix of KSR2 and residue A879 critical for this interaction (i.e., A879L mutation disrupts this interaction). Structure alignment of BRAF KD and KSR2 KD (RMSD, 2.3 A) showed that BRAF R662 would be equivalent to KSR2 A879. (14D) Structure of the wTvlEKI-v^BRAF heterodimer highlighting two orientations of the R662 side chain based on ^WTMEKI-^WTBRAF dimmer co-crystal structure (lighter gray) vs. the vemurafenib-bound ^V600EBRAF crystal structure (medium light gray). The interacting R662 side chain orientation was used to model a putative MEK-BRAF heterodimer interface; R662 was nominated for mutagenesis and additional studies.

[0029] Figures 15A-15B. Related to Figure 7. ERKi-mediated suppression of rebound phospho-ERK unleashed by acute BRAFi and MEKi withdrawal. (15A) M249 DDR4/5 sublines were either cultured with ("on") or without ("off') double drugs (BRAFi, 1 μΜ

vemurafenib; MEKi, 1 μΜ selumetinib). Cultures off double drugs were treated with ERKi (SCH772984) at the indicated concentrations for 1 hours. Western blot analysis of the indicated targets; TUBULIN, loading control. (15B) A low dose of ERKi (0.1 μΜ) was able to suppress p-ERK rebound unleashed by acute BRAFi+MEKi withdrawal over time. An example of Western blots shown for M249 DDR4.

[0030] Figures 16A-16B. Related to Figure 7. (Fig. 16A) Regression of disease progressive melanomas after discontinuation of combined BRAF/MEK targeted therapy. Cases of observed tumor regression subsequent to therapy cessation due to acquired drug resistance among evaluable patients (defined as patients with at least a partial objective response, without intervening treatment after disease progression and cessation of therapy, and with available radiological scans after withdrawal of BRAF and MEK inhibitors; n = 6, Vanderbilt; n = 8, UCSF; n = 1 , UCLA; total n = 15; Table 4). The majority of DP tumors followed continued to progress, with or without tumor growth deceleration, while off treatment. Pt, patient; Tx, treatment; wks, weeks; DP, disease progression. White arrows point to tumors with measurements at the bottom of each CT image. (Fig. 16B) Disease progressive melanomas with tumor growth rate deceleration after discontinuation of single-agent BRAF targeted therapy. Cases of tumor growth rate reduction following discontinuation of BRAF inhibitor therapy due to acquired drug resistance. Tumor growth rates (TGR) were measured as a percentage change (in the greatest diameter of the tumors indicated by the white arrows) per week (wk). TGRs of indicated tumors are shown from all three patients whose melanomas (i.e., target lesions) displayed an overall decelerated TGR (sum of the greatest diameters) (total evaluable patients n = 16; Table 4). No tumor was observed to regress (i.e., with a negative TGR) after vemurafenib withdrawal (range of observation interval without treatment = 34-100 days).

DETAILED DESCRIPTION

[0031] The present invention is based on the discovery of mechanisms of acquired resistance to combined therapy with B-RAF and MEK inhibitors. This discovery enables the

identification of a subset of melanoma patients who respond and subsequently relapse via the described mechanisms. The invention also provides for implementation of a more effective treatment strategy to manage a specific subset of melanoma patients relapsing on and/or developing resistance to combination therapy.

[0032] Definitions

[0033] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

[0034] As used herein, "B-RAF inhibitors" refers to drugs that target an acquired mutation of B-RAF that is associated with cancer, such as ^V600EB-RAF. Representative examples of such a B-RAF inhibitor include PLX4032/vemurafenib or other similar agents, such as

GSK2118436/dabrafenib.

[0035] As used herein, " ^V600E -RAF refers to B-RAF having valine (V) substituted for by glutamate (E) at codon 600. Similar nomenclature is used to indicate other amino acid substitutions or deletions ("del").

[0036] As used herein, "MAPK/ERK kinase (MEK)" refers to a mitogen-activated protein kinase also known as microtubule-associated protein kinase (MAPK) or extracellular signal- regulated kinase (ERK).

[0037] As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.

[0038] Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).

[0039] As used herein, "a" or "an" means at least one, unless clearly indicated otherwise.

[0040] Methods For Identifying Double Drug Resistant Melanoma

[0041] Methods described herein are performed using clinical samples or biopsies derived from patients or short-term culture derived from same. The methods guide the clinician in stratifying patients for sequential treatment strategies with alternative drug(s) or withdrawal and/or intermittent drug therapy.

[0042] In one embodiment, the invention provides a method of predicting or detecting the development of acquired resistance to therapeutic effects of combined B-RAF/MEK inhibitor therapy in a patient suffering from cancer, the method comprising:

(a) assaying a sample obtained from the patient for a measure of combined B- RAF/MEK inhibitor therapy resistance, wherein the measure of resistance is selected from:

V^600EBRAF ultra-amplification (e.g., more than 50 copies, or more than 60 copies, or more than 70 copies, such as about 74 copies);

(2) LOF ^F127VPTEN expression;

(3) homozygous PTEN deletions;

(4) ^G12RNRAS with selective mutant allele amplification;

(5) homozygous CDKN2A deletions (as shown in Table 2);

(6) concurrent heterozygous ^Q61KNRAS with homozygous CDKN2A deletion;

(7) LOF PTEN mutation;

(8) ^V600EBRAF amplification concurrent with homozygous CDKN2A deletion;

(9) ^V600EBRAF amplification concurrent with DUSP4 deletion;

(10) ^V600EBRAF amplification concurrent with MEK1 and MEK2 mutations as

described and tested in Figures 5, 6, 12 and 13.

(11) homozygous CDKN2A deletion concurrent with homozygous PTEN deletion;

(12) hemizygous PIK3R1 deletion; and

(13) any of the genetic profiles listed in Table 2 as associated with double drug disease progression (DD-DP);

(b) selecting samples that exhibit a measure of resistance identified in (a); and (c) identifying a patient whose sample was selected in (b) as susceptible to developing resistance to combined B-RAF/MEK inhibitor therapy.

[0043] Examples of the assaying include, but are not limited to, targeted sequencing, realtime RT-PCR, Sanger sequencing and/or whole exome sequencing. Primers for use in these assays are provided in Example 2 below. Kits comprising these primers, or subsets of these primers for targeting mutations identified herein, are also provided by the invention. Such kits may optionally include a nucleotide sequence for use as a positive control in methods of the invention. Also optionally included in the kits are polymerizing enzymes, deoxynucleotide triphosphates (A, G, C, T), and containers for housing each of these components of the kit.

[0044] The combined B-RAF/MEK inhibitor therapy can comprise, for example, treatment with a B-RAF inhibitor selected from vemurafenib and dabrafenib, and/or treatment with a MEK inhibitor selected from trametinib, selumetinib, cobimetinib, and binimetinib. Typically, the sample is selected from tissue, bodily or biologic fluid, such as blood, tumor biopsy, spinal fluid, and needle aspirate. The method can be performed prior to treatment with combined B- RAF/MEK inhibitor therapy, after treatment with combined B-RAF/MEK inhibitor therapy, during disease progression or clinical relapse on combined B-RAF/MEK inhibitor therapy, or after suspension of combined B-RAF/MEK inhibitor therapy. In a typical embodiment, the cancer is melanoma.

[0045] In some embodiments, the method further comprises identifying in a patient who is susceptible to developing or has developed resistance to combined B-RAF/MEK inhibitor therapy the form(s) of resistance and method(s) to counter such resistance. The method can, optionally, further comprise treating the patient with intermittent dosing of combined B-RAF and MEK inhibitors. Methods to counter resistance driven by described mechanisms include (but are not limited to): combinations based on BRAF and MEK inhibitors with the additions of: Inhibitors of the PI3K-AKT-mTORC pathway; Inhibitors downstream of the MAPK pathway such as CDK4/6 inhibitors; Inhibitors that block MEK activation by RAF; Inhibitors that block BRAF and MEK interactions; Inhibitors that block RAF dimerization; and Inhibitors that block RAS functions.

[0046] Examples of inhibitors of the PI3K-AKT-mTORC pathway include, but are not limited to, PI3K p110b inhibitor GSK 2636771 , AKTi GSK690693, and PI3K/mTOR dual inhibitor GSK2126458. Examples of inhibitors downstream of the MAPK pathway include, but are not limited to, CDK4/6 inhibitors, e.g., palbocuclib, LEE011 and inhibitors that block MEK activation by RAF. Newer MEK inhibitors target MEK catalytic activity and also impair its reactivation by CRAF, either by dismpting RAF-MEK complexes or by interacting with Ser 222 to prevent MEK phosphorylation by RAF. See Lito P, et al., Cancer Cell. 2014 May 12;25(5):697-710. doi:

10.1016/j.ccr.2014.03.011. Epub 2014 Apr 17. PMID: 24746704. [0047] Examples of inhibitors that allosterically block MEK1/2 activities include, but are not limited to, e.g., trametinib, cobimetinib, and selumetinib. Experimental compounds are available that block BRAF and MEK interactions. Examples of inhibitors that block RAF dimerization include, but are not limited to, experimental compounds and LY3009120.

Examples of inhibitors that block RAS functions include, but are not limited to, KRAS G12C inhibitor 6.

[0048] The invention additionally provides a method of preventing or suppressing acquired resistance to combined B-RAF/MEK inhibitor therapy in a patient. In one embodiment, the method comprises administering to the patient a therapeutic agent selected from the group consisting of:

(a) a suppressor of C-RAF kinase activity;

(b) an inhibitor of activation of MEK1 or MEK2 by C-RAF; and

(c) an inhibitor of activation of ERK1 or ERK2 by MEK1 and MEK2.

[0049] In one embodiment, the therapeutic agent is selected from the group consisting of: Omni-RAF, CRAF and RAF paradox breakers, and ERK inhibitors. In another embodiment, the therapeutic agent is CCT196969, CCT241161, PLX3397, PLX7904 and/or SCH442984.

[0050] Therapeutic and Prophylactic Methods

[0051] The invention further provides a method of treating a patient having cancer, or who may be at risk of developing cancer or a recurrence of cancer. In a typical embodiment, the patient has melanoma. In one embodiment, the melanoma is a B-RAF-mutant melanoma. The cancer can be melanoma or other cancer associated with B-RAF mutation, such as, for example, ^V600EB-RAF. Patients can be identified as candidates for treatment using the methods described herein. Patients are identified as candidates for treatment on the basis of exhibiting one or more indicators of resistance to B-RAF inhibitor therapy. The treatment protocol can be selected or modified on the basis of which indicators of resistance to B-RAF inhibitor therapy are exhibited by the individual patient.

[0052] The patient to be treated may have been initially treated with conventional B-RAF inhibitor therapy, or may be a patient about to begin B-RAF inhibitor therapy, as well as patients who have begun or have yet to begin other cancer treatments, including treatment with ERK inhibitors, for example. Patients identified as candidates for treatment with one or more alternative therapies can be monitored so that the treatment plan is modified as needed to optimize efficacy.

[0053] Examples of alternative therapy include, but are not limited to, intermitting dosing with combined B-RAF/MEK inhibitor therapy, augmenting B-RAF/MEK inhibitor therapy with at least one additional drug. The additional drug can include a MAPK/ERK kinase (MEK) inhibitor, such as PD0325901 , GDC0973, GSK1120212, and/or AZD6244. In one

embodiment, the alternative therapy comprises suspension of vemurafenib therapy.

[0054] In one embodiment, the alternative therapy comprises administering to the patient a MEK inhibitor, optionally in conjunction with vemurafenib therapy, or an inhibitor of the MAPK pathway (RAF, MEK, ERK) in conjunction with an inhibitor of the RTK-PI3K-AKT-mTOR pathway. Examples of MEK inhibitors include, but are not limited to PD0325901 , GDC0973, GSK1120212, and/or AZD6244§. Examples of inhibitors of the RTK-PI3K-AKT-mTOR pathway include, but are not limited to BEZ235, BKM120, PX-866, and GSK2126458.

[0055] In a typical embodiment, the invention provides a method of preventing or suppressing acquired resistance to combined B-RAF/MEK inhibitor therapy in a patient. The method comprises administering to the patient a therapeutic agent selected from the group consisting of: a suppressor C-RAF kinase activity, and an inhibitor of activation of MEK1 or MEK2 by C- RAF or ERK1 or ERK2 by MEK1 and MEK2. Representative examples of a therapeutic agent include, but are not limited to: Omni-RAF, CRAF and RAF paradox breakers (e.g.,

CCT196969, CCT241161 , PLX3397, and PLX7904) and ERK inhibitors (e.g., SCH772984).

[0056] The method of preventing or suppressing acquired resistance to combined B- RAF/MEK inhibitor therapy in a patient can be implemented, for example, when such resistance results from MAPK pathway reactivation. MAPK pathway reactivation can be measured by detecting rebound phospho-ERK levels in response to acute BRAF and MEK inhibitor withdrawal or speed of recovery of phospho-ERK levels after a single dose of BRAF and MEK inhibitor treatment.

[0057] Treatment includes prophylaxis and therapy. Prophylaxis or therapy can be accomplished by a single administration or direct injection, at a single time point or multiple time points to a single or multiple sites. Administration can also be nearly simultaneous to multiple sites. Patients or subjects include mammals, such as human, bovine, equine, canine, feline, porcine, and ovine animals. The subject is preferably a human. In a typical embodiment, treatment comprises administering to a subject a pharmaceutical composition of the invention.

[0058] A cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor. Pharmaceutical compositions may be administered either prior to or following surgical removal of primary tumors and/or treatment such as

administration of radiotherapy or conventional chemotherapeutic drugs.

[0059] Administration and Dosage

[0060] The compositions are administered in any suitable manner, often with

pharmaceutically acceptable carriers. Suitable methods of administering treatment in the context of the present invention to a subject are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0061] The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial therapeutic response in the patient over time, or to inhibit disease progression. Thus, the composition is administered to a subject in an amount sufficient to elicit an effective response and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease. An amount adequate to accomplish this is defined as a "therapeutically effective dose."

[0062] Routes and frequency of administration of the therapeutic compositions disclosed herein, as well as dosage, will vary from individual to individual as well as with the selected drug, and may be readily established using standard techniques. In general, the

pharmaceutical compositions may be administered, by injection (e.g., intracutaneous, intratumoral, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. In one example, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster treatments may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. In one embodiment, 2 intradermal injections of the composition are administered 10 days apart.

[0063] A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored using conventional methods. In general, for pharmaceutical compositions, the amount of each drug present in a dose ranges from about 100 pg to 5 mg per kg of host, but those skilled in the art will appreciate that specific doses depend on the drug to be administered and are not necessarily limited to this general range. Likewise, suitable volumes for each administration will vary with the size of the patient.

[0064] In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients.

[EXAMPLES

[0065] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention. [0066] Example 1: Tunable-Combinatorial Mechanisms of Acquired Resistance Limit the Efficacy of BRAF/MEK Cotaraetina but Result in Melanoma Drug Addiction

[0067] Combined BRAF/MEK targeted therapy improves upon BRAF inhibitor (BRAFi) therapy but is still beset by acquired resistance. This Example shows that melanomas acquire resistance to combined BRAF/MEK inhibition by augmenting or combining mechanisms observed in single-agent BRAFi resistance. Double-drug resistant melanomas tuned up (e.g., ^V600EBRAF, ^G12RNRAS) or down (e.g., PTEN, CDKN2A) resistance driver genes or combined individual mechanisms (e.g., ^V600E BRAF amplification+^F129LMEKf mutation,

Q^61RNRAS+^FZ71VPTEN). These resistance-associated genetic configurations impact survival signaling both quantitatively and qualitatively. Supra-physiologic levels of ^V600EBRAF, resulting from gene ultra-amplification, dimerized with and activated CRAF. Also, MEK1 mutants enhanced interaction with over-expressed ^V600EBRAF. Importantly, melanoma cell lines selected for resistance to BRAFi+MEKi, but not those to BRAFi alone, displayed robust drug addiction, a potentially exploitable therapeutic opportunity.

[0068] Significance

[0069] The understanding that BRAF mutant melanomas frequently acquire BRAFi resistance via MAPK pathway reactivation has guided the development of combined BRAF/MEK targeted therapy, an approach recently approved by the FDA. The surprising discovery described herein, that acquired resistance to BRAF/MEK co-targeting is driven by highly tunable-combinatorial mechanisms of resistance, underscores the intrinsic limitation of dual MAPK pathway targeting. Mechanistic studies highlight ^V600EBRAF-^WTCRAF and ^V600EBRAF- ^MUTMEK interactions as a basis for ERK-reactivation. Additionally, this Example demonstrates that melanoma cells with acquired BRAFi+MEKi resistance are exquisitely sensitive to acute drug withdrawal (vs. those with acquired resistance to BRAFi alone). Exploiting melanoma addiction to BRAFi+MEKi for therapeutic gain can be addressed via intermittent drug dosing, in the clinic (SWOG/CTEP S1320).

[0070] EXPERIMENTAL PROCEDURES

[0071] Patients, Tumor Samples, and Genomic Analysis

[0072] Melanoma tissues and patient-matched normal tissues were collected with the approval of institutional review boards at University of California, Los Angeles; University of California, San Francisco; and Vanderbilt-lngram Cancer Center and informed consents of each patient. Patients were enrolled in GlaxoSmithKline or Roche/Genetech clinical trials or treated per standard clinical management. We evaluated 45 tumor samples (27 DD-DP, 4 DP, and 14 baseline or early on-treatment melanoma biopsies) from 14 patients who were either treated with BRAFi+MEKi upfront or with this combination after progression on BRAFi. In each tumor, genetic mechanisms (excluding PI3K-PTEN-AKT genetic hits) known to confer clinical resistance to BRAFi were detected by gDNA qPCR and/or Sanger sequencing.

Twenty-three baseline and DD-DP tumors from seven patients along with normal tissues were WES analyzed to detect somatic alterations that are in the MAPK and PI3K-PTEN-AKT pathways and that are specific to drug-resistant tumors.

[0073] Pair-end sequences with read length of 2x100 base pairs using the lllumina

HiSeq2000 platform were generated. SNVs, insertion-deletions (INDELs), and CNVs were analyzed and visualized as described previously (Shi et al., 2014).

[0074] Targeted Sequencing, Copy-Number Quantification, and WES of Cell Lines

[0075] BRAF.NRAS, and DUSP4cDNAIevels were quantified by real-timeRT-PCRusing TUBULIN and GAPDH levels for normalization. Relative expressions were calculated using the delta-Ct method. BRAF, NRAS, and DUSP4 gDNA relative copy numbers were quantified by real-time PCR with total gDNA content estimated by assaying the b-globin gene in each sample. All primer sequences are listed in Example 2 below. Sanger sequencing was performed using purified PCR via BigDye v1.1 (Applied Biosystems) in combination with a 3730 DNA Analyzer (Applied Biosystems). WES of M249 triple cell lines were analyzed for shared and distinct genetic alterations and their phylogenetic relationship.

[0076] Cell Culture, Constructs, Infections, and Transfections

[0077] All cell lines were maintained in DMEM with 10% heat-inactivated fetal bovine serum, 2 mmol/l glutamine in a humidified 5% C02 incubator, with the addition of 10 ng/ml doxycycline and or puromycin, when applicable. Stocks and dilutions of

PLX4032/vemurafenib (Plexxikon), AZD6244/selumetinib (Selleck Chemicals), and

SCH772984 (Merck) were made in DMSO. Cell proliferation experiments were performed in a 96-well format (five replicates per sample), drug treatments were initiated 24 hr postseeding for 72 hr, and cell survival was quantified using CellTiter-GLO assay (Promega). Clonogenic assays were performed by plating cells at single-cell density in six-well plates with fresh media and drug replenished every 2 days. Colonies were fixed in 4% paraformaldehyde and stained with 0.05% crystal violet. shBRAF, shCRAF, shPTEN, and shNRAS were subcloned into the lentiviral vector pLL3.7; shDUSP4/pLK0.1 vectors were obtained commercially (Dharmacon). All WT and mutant MEK1 and BRAF constructs were epitope tagged and subclonedinto the doxycycline-repressible lentiviral vector pLVX-Tight-Puro (Clontech Laboratories). Viral supernatants were generated by third-generation lentiviral packaging using HEK293T cells. HEK293T cells were transfected using BioT (Bioland).

[0078] Protein Detection, Interaction, and Structure

[0079] Cell lysates were made in radioimmunoprecipitation assay buffer (Sigma) for direct western blotting or in a PNE buffer (PBS:H20 at 1 : , 0.5% Nonidet P-40, 5mMEDTA, and5% glycerol) for immunoprecipitation, with both buffers supplemented with protease (Roche) and phosphatase (Santa Cruz Biotechnology) inhibitor cocktails. Western blots and

immunoprecipitations were performed using the following antibodies: p-ERKI/2 (T202/Y204), P-MEK1/2 (S217/221), p-AKT (T308), p-CRAF (S338), total ERK1/2, MEK1/2, MEK1 , MEK2, AKT, CRAF, DUSP4, and HA (Cell Signaling Technology); TUBULIN and FLAG (Sigma); BRAF (F-7), BRAF (C-19), p-MEK1 (T291), and p-MEK1 (S222) (Santa Cruz); and p-MEK2 (S226) (United States Biological). Western blot quantification was performed using NIH ImageJ. The 3D structures of MEK1 (3EQC) and PTEN mutants were modeled by the I- TASSER online server. Modeling the ^V600EBRAF-^MUTMEK1 dimer interface was based on the crystal structure of the ^WTBRAF-^WTMEKI dimer (4MNE); the MEK1-KSR2 dimer (2Y4I); and the asymmetric, vemurafenib-bound ^V600EBRAF dimer (3G07). Protein structures were visualized using PyMol (DeLano Scientific).

[0080] The Sequence Read Archive accession number for the exome sequence data reported in this Example is SRP049746.

[0081] Supplemental Information providing greater details includes six figures, four tables, and one movie and can be obtained in connection with the publication of this article as Cancer Cell 27, 1-17, February 9, 2015 using dx.doi.org/10.1016/j.ccell.2014.11.018.

[0082] RESULTS

[0083] Genetic Alterations Underlying Acquired Resistance to BRAF/MEK Cotargeting in Melanoma

[0084] We assembled melanoma tissues with acquired resistance to BRAFi+MEKi

(abbreviated as DD-DP for double-drug disease progression) (n = 28 DD-DP tumors, each with patient-matched baseline tumors) from patients (n = 15) treated under two distinct clinical scenarios (Figure 1A): (1) upfront BRAFi+MEKi (dabrafenib+trametinib or

vemurafenib+cobimetinib) in patients (n = 10) who were naive to treatment with either BRAFi or MEKi and (2) BRAFi+MEKi (vemurafenib+cobimetinib) in patients (n = 5) who had previously responded to but progressed on BRAFi (vemurafenib) alone (Table 1). We then analyzed known mechanisms of acquired BRAFi resistance in the MAPK pathway by sequencing the most pertinent exons of BRAF, NRAS, KRAS, MEK1, and MEK2 and performing BRAF copy-number analysis (Table 2). Sixteen of 28 DD-DP tumors, along with their patient-matched baseline tumors and normal tissues (n = 7), were whole exome sequenced and analyzed for MAPK and PI3K-PTEN-AKT pathway alterations as reported previously (Shi et al., 2014) (Table 2). In 19 of 28 (68%) DDDP tumors, we detected known mechanisms of acquired BRAFi resistance in the two core resistance pathways. These included eight DD-DP tumors harboring ^V600EBRAF amplification, four harboring NRAS activating mutations, one harboring a KRAS activating mutation, eight harboring CDKN2A deletions, three harboring PTEN loss-of-function (LOF) mutation (a substitution resulting in F127V; Figure 9) or deletions, and one harboring a PIK3R1 deletion. In contrast to the same alterations detected in the context of resistance to BRAFi monotherapy (Shi et al., 2014; Van Allen et al., 2014), those associated with acquired BRAFi+MEKi resistance were notable for augmented gene dosage changes, e.g., ^V600EBRAF ultra-amplification with 74 or 88 copies (Figure 1 B; Table 2), LOF F127VPTEN mutation or homozygous PTEN deletions (Figure 1C), ^G12RNRAS with selective mutant allele amplification (Figures 1D and 1 E), and homozygous CDKN2A deletions (Table 2). There were examples suggesting combinatorial mechanisms, e.g., concurrent heterozygous Q61 KNRAS with homozygous CDKN2A deletion and LOF PTEN mutation; ^V600EBRAF amplification concurrent with homozygous CDKN2A deletion or hemizygous DUSP4 deletion (with related ^V600EBRAF up-expression and DUSP4 down- expression; Figures 1 F-1 H); and homozygous CDKN2A deletion concurrent with

homozygous PTEN deletion and hemizygous PIK3R1 deletion. Thus, genetic analysis of melanomas progressing on BRAFi+MEKi revealed a prevalence of mechanisms of acquired BRAFi resistance, but these genetic alterations often occurred in greater magnitudes or in combinations.

[0085] Table 1 : Clinical characteristics of patients and their responses to BRAF + MEK

VICC, Vanderbilt Ingram Cancer Center

UCLA, University of California, Los Angeles

UCSF, University of California, San Francisco

Dabra, dabrafenib (orally dosed twice a day)

Trame, trametinib (orally dosed once a day)

Vemu, vemurafenib (orally dosed twice a day)

GDC0973 (cobimetinib, orally dosed once a day)

Patients #7-11 were treated with BRAFi+MEKi as their melanoma progressed on BRAFi monotherapy (all other patients were treated upfront with BRAFi+MEKi). For these patients, PFS refers to

BRAFi+MEKi treatment.

N/A, not available; LN, lymph node; SC, subcutaneous

[0086] Table 2: Summary of genetic alterations detected in core resistance pathway.

melanoma) with whole-exome sequence data.

Box in darker grey, data not available.

- , event not observed.

* Copy number gains shown as fold increase (relative to baseline) and copy number in DD-DP.

*^"Hits" in the MAPK pathway, which included the genes directly interacting with the RAS, RAF, MEK and ERK genes in the KEGG's MAPK signaling pathway (KEGG ID: hsa04151). Copy number

**^'Hits" in the PI3K-PTEN-AKT pathway, which included genes directly interacting with PI3K,

PTEN and AKT in the KEGG's PI3K-AKT signaling pathway (KEGG ID: hsa04151). RAF1

(CRAF) was excluded as it is one of the MAPK signaling pathway's genes.

AMP, amplification or copy number increase.

DEL, copy number decrease.

Homo, hemozygous.

Hem, hemizygous.

[0087] BRAFi-Resistant Melanoma Rapidly Upregulates Resistance Mechanisms Individually or Combinatorially to Overcome BRAF/MEK Inhibitors

[0088] To further understand acquired BRAFi+MEKi resistance in melanoma underlying the two aforementioned clinical contexts, we generated isogenic human ^V600EBRAF melanoma cell lines using treatment regimens mimicking each clinical context. In the sequential resistance model, we took those isogenic sublines with acquired BRAFi (vemurafenib) resistance (single-drug resistance or SDR), via clinically validated mechanisms such as NRAS mutation (M249R4) (Nazarian et al., 2010), ^V600EBRAFalternative splicing (M397R) (Shi et al., 2012b), or amplification (M395R) (Shi et al., 2012b), and generated further sublines with BRAFi+MEKi (vemurafenib+selumetinib) or double-drug resistance (DDR). In the upfront BRAFi+MEKi resistance model, we took the same set of parental (P), drug-naive melanoma cell lines and treated them at the outset with BRAFi+MEKi until we generated sublines with DDR (Figure 2A). The cell subpopulations were exposed to similar increments of inhibitor concentrations, with the duration at each inhibitor concentration dictated by successful population doubling within 3-4 days. When the timecumulative doses to reach the full DDR phenotype (defined as 2 mM BRAFi+MEKi) were compared between these two models, it was clear that the development of DDR from SDR was much faster than DDR directly from P lines (Figure 2A). This observation is consistent with the hypothesis that preexisting mechanisms of BRAFi resistance could be readily augmented or tuned up to confer resistance to BRAFi+MEKi.

[0089] To assess this hypothesis, we examined the SDR versus SDRDDR isogenic pairs of cell lines for alterations in the preexisting, defined mechanisms of BRAFi resistance (Figures 2B-2E). We showed that the M397 SDR/DDR progression was associated with a dramatically upregulated level of alternatively spliced ^V600EBRAF mRNA (Figure 2B). Moreover, the M395 SDR/ DDR progression resulted in further ^V600EBRAF amplification along with RNA up- expression. The M249 SDR/DDR progression upregulated mutant NRAS mRNA levels without genomic DNA (gDNA) copy-number gain (Figures 2C and 2D). Accordingly, at the protein expression level (Figure 2E), M249 SDRDDR expressed an increased NRAS level; M397 SDR-DDR upregulated the level of a truncated p61 ^V600EBRAF; and M395 SDR-DDR upregulated ^V600EBRAF expression further (all relative to isogenic SDR sublines). Moreover, full-length ^V600EBRAF overexpression (in M395 SDR or SDR-DDR) was associated with extensive phospho (p)-CRAF levels (versus their P line). Thus, common mechanisms of acquired BRAFi resistance are highly tunable by either genetic or nongenetic means, and augmentation or combination of such molecular alterations readily confers resistance to BRAFi+MEKi.

[0090] We then tested whether specific examples of gene dosage augmentation or concurrent genetic alterations from the exomic analysis of paired melanoma tissues would augment BRAFi+MEKi resistance in cell line models. Parallel to the mutant NRAS amplifications detected in both DD-DPs of patient 9 (Figures 1 D and 1 E), M249 SDR-DDR up-expressed mutant NRAS (albeit via a nonmutational mechanism) (versus P or SDR) (Figures 2A and 2C-2E). NRAS knockdown (Figure 2F) restored BRAFi sensitivity to M249 SDR, as would be expected, but it also strongly restored BRAFi+MEKi sensitivity to M249 SDRDDR in both short- and long-term (Figures 2G and 2H) survival assays, indicating that overexpression of mutant NRAS drove DDR. To engineer a DDR cell line mimicking Q61 KNRAS

heterozygosity+F271VPTEN/K197*PTEN compound heterozygous mutations (DD-DP of patient 6; Figure 1C), we took advantage of the PTEN-expressing, Q61 KNRAS-driven M238 SDR subline (Figure 2I), which was derived from its ^V600EBRAF P line by incremental exposures to increasing doses of BRAFi, and stably introduced small hairpin (sh)PTEN. We showed that PTEN knockdown in M239 SDR increased the p-AKT level (Figure 2J) and resistance to BRAFi+MEKi (Figure 2K), indicating that each resistance mechanism

(Q61 KNRAS and PTEN loss) quantitatively contributed to DDR. Moreover, given that ^V600EBRAF amplification concurred with hemizygous DUSP4 deletion (DD-DP1 and DD-DP2 of patient 11), we tested whether DUSP4 knockdown (Figure 2L) could confer DDR to the M395 SDR subline, which acquired BRAFi resistance via ^V600EBRAF amplification. As seen in Figure 2M, M395 SDR was moderately cross-resistant to BRAFi+MEKi treatments, but loss of DUSP4 expression augmented DDR.

[0091] Clonal Analysis Detects Alternative Genetic Configurations in BRAFi+MEKi Resistance Associated with MAPK Reactivation

[0092] Previous results indicate that once subclones with specific BRAFi resistance mechanisms have attained clonal dominance, overcoming BRAFi resistance with the added MEKi is at best an uphill battle. We then sought to understand the underlying mechanism(s) of resistance to upfront BRAFi+MEKi (vemurafenib+selumetinib). A polyclonal DDR subline derived from M249 harbored both mutant BRAF ultra-amplification and an MEK1 mutation (F129L). ^F129LMEK1 had previously been uncovered in a colorectal subline bred to acquire selumetinib resistance (Wang et al., 2011). To understand the individual contributions of ^V600EBRAF amplification and MEK1 mutation to the DDR phenotype, we retreated the M249 P with increments of BRAFi+MEKi but derived two single-cell-derived M249 DDR subclones, DDR4 and DDR5. In contrast to M249 P, both DDR4 and DDR5 were highly resistant to the growth-inhibitory effect of BRAFi+MEKi in 3-day 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assays (Figure 3A). In fact, the apparent "growth stimulation" of DDR4 and DDR5 by BRAFi+MEKi treatment was due to a relative loss of their viability in the absence of optimal concentrations of the inhibitors. This "drug addiction" phenomenon was even more profound in long-term clonogenic assays (see Figure 4).

SCH772984, an ERK inhibitor (ERKi) and an analog of which is being tested clinically, was inefficient to inhibit the growth of DDR4 or DDR5 by itself but was highly active against M249 P (Figure 3A). In fact, low concentrations of SCH772984 rescued DDR4 and DDR5 from drug addiction, suggesting that suboptimal ERKi dosing to overcome DDR may paradoxically perpetuate DDR fitness. In contrast, ERKi restored BRAFi+MEKi sensitivity to DDR4 and DDR5, consistent with MAPK pathway reactivation as the major mechanism of acquired resistance to upfront BRAFi+MEKi. This was corroborated by analyzing the MAPK pathway status (p-ERK levels) in the M249 triplet (Figure 3B). After plating for 16 hr without both inhibitors, the triplet cell lines were treated with BRAFi+MEKi (1 hr) at increasing

concentrations (Figure 3B) or with BRAFi+MEKi (1 mM) for increasing durations (up to 72 hr) (Figure 10A). Western blot analysis showed that DDR4 and DDR5, compared to M249 P, displayed higher baseline and inhibitor- treated p-ERK levels as well as faster p-ERK recovery in the continued presence of BRAFi+MEKi. Monitoring further upstream for p-MEK and downstream for p-RSK (T573) levels revealed a similarly rapid recovery of the MAPK pathway (Figure 10B).

[0093] Consistent with the BRAF protein levels (Figure 3B), we found that DDR4 harbored ^V600EBRAF ultra-amplification (47.4-fold or >160 copies), while DDR5 harbored low copy- number ^V600EBRAF gain (4.6-fold or 20 copies) along with ^F129LMEK1 (Figures 3C and 3D). ^V600EBRAF copy-number gains quantified by gDNA quantitative (q)PCR were corroborated by Sanger sequencing, which showed a BRAF mutant-to-WT ratio of 2: 1 in the P line (or about three BRAF copies) and apparent ^V600EBRAF homozygosity in DDR4 and DDR5 resulting from selective ^V600EBRAF amplification (Figure 3D). Moreover, whole exome sequence (WES) analysis of the M249 triplet cell lines confirmed that the DDR-associated altered mutant or variant allelic frequencies (MAFs) of ^V600EBRAF and ^F129LMEK1 (Figure 3E) were likely due to mutant allele-selective copy-number gains (Figure 3F). WES analysis also detected a low ^F129LMEK1 MAF (4%) in the P polyclonal line, suggesting preexistence of this drug-resistant subclone (Figure 3E). In addition, copy-number variation (CNV) analysis revealed distinct BRAF amplicons in M249 DDR4 versus DDR5, suggesting convergent evolution (Figure 3F, top). Since the concurrence of ^V600EBRAF amplification and ^F129LMEK1 in M249 DDR5 could be selected by distinct inhibitor concentrations, we derived two additional M249 DDR sublines (M249 DDR2 and M249 DDR3) by treatments from the outset with a higher concentration of BRAFi+MEKi (0.5 mM). In a pattern suggestive of convergent evolution, both M249 DDR2 and DDR3 displayed low copy-number gains of both ^V600EBRAF and ^F129LMEK1 (Figures 10C- 10E).

[0094] Using a ^WTBRAF cell line, human embryonic kidney 293T (HEK293T) cells, we then tested the impact of MEK mutants associated with MAPK inhibitor (MAPKi) resistance on cellular substrate levels (i.e., p-ERK) and the p-ERK half-maximal inhibitory concentration (IC50) of MEKi (Figures 10F and 10G). We overexpressed ^F129LMEK1 , ^C121SMEK1 (which confers BRAFi resistance) (Wagle et al., 2011), and several MEK mutants (^Q56pMEK1, ^{K59 del}MEK1 , and ^E203KMEK1) associated with clinical resistance to MAPK targeting and compared their impacts on baseline p-ERK levels as well as the sensitivities of p-ERK to MEKi

(selumetinib). Although overexpression of these MEK1 mutants (versus ^WTMEKI) variably increased the baseline p-ERK level, their cellular p-ERK IC50 value to MEKi did not differ appreciably, arguing against allosteric MEKi binding defect as the shared mechanism of action of MEK mutants. Their concurrence with ^V600EBRAF amplification argues for a possible cooperative biochemical mechanism of resistance.

[0095] To further understand the impact of ERKi on survival of DDR4 and DDR5 cells (Figure 3A), we withdrew DDR4 and DDR5 (16 hr off) from BRAFi+MEKi and then treated them with either ERKi (1 hr) alone or BRAFi+MEKi+ERKi (1 hr) (Figure 3G). ERKi alone was ineffective at suppressing the p-ERK rebound following double-drug withdrawal (Figure 3G). However, once BRAFi+MEKi was reintroduced, additional treatment with ERKi was highly effective in suppressing the p-ERK levels (Figure 3G). Thus, ERKi treatment alone of some melanoma cells previously selected for resistance by BRAFi+MEKi would be ineffective unless very high ERKi doses were delivered, which is unlikely achievable clinically. Thus, clonal M249 DDR4 and DDR5 melanoma sublines harbor salient but distinct genetic alterations that represent tunable and combinatorial modes of resistance to BRAFi+MEKi reversible by combining ERKi.

[0096] Distinct Mechanisms of Resistance Driven by ^V600EBRAF Ultra-amplification or ^V600EBRAF Amplification+^F129LMEK1

[0097] Earlier, we noted a robust upregulation of p-CRAF in the M395 SDR and SDR-DDR sublines that harbor ^V600EBRAF amplification (Figure 2E). Hence, we probed the p-CRAF levels in the M249 triplet lines. DDR4 and DDR5, freshly treated with BRAFi+MEKi (1 hr), displayed robust elevated p-CRAF levels (DDR4 > DDR5 » P; Figure 3H). Upregulated p- CRAF levels in DDR4 and DDR5 did not require the continued presence of both inhibitors, as their withdrawal for up to 20 hr after an overnight (16 hr) treatment did not diminish the p- CRAF levels (Figure 10H). We hypothesized that this strong CRAF upregulation in DDR4 (and a weaker upregulation in DDR5) may be driven by supraphysiologic ^V600EBRAF overexpression, the degree of which positively correlated with that of p-CRAF upregulation (Figures 3B, 3H, and 4A). To test this hypothesis, we knocked down BRAF levelsin DDR4 and DDR5, with or without BRAFi+MEKi, and we found that BRAF knockdown effectively downregulated p-CRAF levels (Figure 4A). BRAF knockdown also reduced p-CRAF levels in the ^V600EBRAF-amplified M395 SDR-DDR subline (Figure 2B; Figure 11 A). We also knocked down CRAF directly (Figure 4B) and tested the individual contributions of BRAF versus CRAF to the clonogenic (i.e., long-term) growth and survival of the M249 triplet (Figure 4C). As expected, M249 P growth and survival was not sensitive to CRAF knockdown but highly sensitive to BRAF knockdown. Consistent with prior short-term assays (Figure 3A), both M249 DDR4 and DDR5 displayed dramatic drug addiction. Importantly, in the presence of both inhibitors, the growth and survival of DDR4 and DDR5 was highly dependent on either CRAF or BRAF, suggesting functional and physical interaction. [0098] To assess whether there are likely additional genetic underpinnings of p-CRAF upregulation (and DDR) in DDR4 and DDR5, we analyzed the phylogenetic relationship of the M249 triplet (Figure 4D) and assessed the genetic alterations shared by DDR4 and DDR5 (Table S3 available online in connection with Moriceau et al., 2015, Cancer Cell 27(2):240- 56). From this WES-based phylogeny, it was apparent that DDR4 and DDR5 single-cell clones represent minor subclones in the P, polyclonal population, since they each harbor a large number of private mutations, which escaped detection in the mixed P population. In fact, the number of shared genetic alterations between DDR4 and DDR5 was exceedingly small (Table S3, see Moriceau et al., 2015, cited above), suggesting that these few alterations (aside from ^V600EBRAF amplification) were unlikely drivers of DDR. As the M249 P majority population does not harbor the DDR4- or DDR5-private mutations, we reasoned that the ability of salient genetic feature shared by DDR2, DDR3, DDR4, and DDR5 (^V600EBRAF amplification) and by DDR2, DDR3, and DDR5 (^F129LMEK1 and its low copy-number gain) to reconstitute DDR (and their biochemical features) would establish sufficiency (in light of necessity established earlier).

[0099] We then directly tested whether supraphysiologic ^V600EBRAF overexpression, mimicking the DDR4 and DDR5 levels, would be sufficient to upregulate p-CRAF levels. We engineered the M249 P to express stably and homogeneously the empty vector, ^F129LMEK1 (Figure 4E), or ^Q56PMEK1 (Figure 4F) (identified in clinical MEKi [Villanueva et al., 2013] or BRAFi+MEKi [Wagle et al., 2014] resistance, respectively), ^V600EBRAF high overexpression, and ^V600EBRAF low overexpression concurrent with a MEK1 mutation. Regardless of double- drug treatment (16 hr) or subsequent withdrawal (8 hr), ^V600EBRAF high overexpression induced a robust DDR4-like p-CRAF level, while ^V600EBRAF low overexpression concurrent with an MEK1 mutation induced a lower, DDR5-like p-CRAF level. Neither vector control nor ^MUTMEK1 alone had any impact on the p-CRAF level. Also, supraphysiologic expression of ^WTBRAF or V600E/R509H_braf (known to disrupt BRAF-CRAF dimerization) in M249 P only marginally upregulated p-CRAF (Figure 4G). However, the M249 P engineered cell lines (versus the spontaneously resistant DDR4 and DDR5 sublines), displayed a slower p-ERK recovery (with or without BRAFi+MEKi; Figures 4E and 4F). This difference (a few hours) was minimal compared to the extremely slow p-ERK recovery observed in the P line (not detectable by 3 days; Figure 10A) and appeared to be due to prior MAPKi exposure or preconditioning, which abolished the small difference in the p-ERK recovery rate between the M249 P engineered lines versus DDR4 and DDR5 (Figure 11 B).

[00100] We then assessed the relative potencies of individual alterations observed in M249 DDR4 and DDR5 to confer BRAFi+MEKi resistance in M249 P using both short-term (Figures 11C and 11 D) and long-term (Figures 4H) survival assays. ^V600EBRAF high overexpression or ^V600EBRAF low overexpression concurrent with an MEK1 mutant (F129L or Q56P) conferred more than one-log (short-term) or two-log (long-term) increases in MAPKi resistance.

Interestingly, preconditioning of the engineered M249 lines conferred double-drug addiction (Figures 11 E and 11F). As was noted previously for DDR4 and DDR5 sublines (Figures 3A and 4C), the double-drug addiction phenotype also exaggerated the apparent DDR phenotype of preconditioned M249 P engineered with each genetic configuration (Figure

11 D). These data together (Figures 4E and 4F; Figures 11 B-11 F) thus suggest a mechanistic link between double drug addiction and p-ERK rebound (see below in Figure 7). Moreover, supraphysiologically expressed V600E/R509^HBRAF, defective in p-CRAF induction (Figure 4G), was also compromised in its ability to resist repeated treatments with BRAFi+MEKi (1 mM, 24 days). ^V600EBRAF low overexpression or MEK1 mutation alone was individually able to confer BRAFi+MEKi resistance, but only to an extent appreciably weaker than achieved by their combination (see growth at 0.1 versus 1.0 mMof drugs at days 15 and 24) (Figure 4H). The combinatorialeffects of overexpressed ^V600EBRAF and MEK1 mutants on promoting the DDR phenotype could also be observed in a different cell line (Figures 11G-11 I). Thus, ERKi- sensitive, acquired resistance to BRAFi+MEKi observed in DDR4 and DDR5 is causally attributable to either supraphysiologic overexpression of ^V600EBRAF or a lower degree of ^V600EBRAF and ^MUTMEK overexpression (Figure 4I; Figure 11). Mechanistically, excess ^V600EBRAF proteins promote dimerization with CRAF and CRAF activation and dependency.

[00101] Next, to dissect mechanistically how ^MUTMEK1 aids overexpressed ^V600EBRAF in establishing a full DDR phenotype, we posited that overexpressed ^V600EBRAF and ^MUTMEK physically and functionally interact in a complex facilitated by (1) the MEK mutant

conformation and (2) a kinase-independent regulatory role of ^V600EBRAF. This complex facilitates MEK phosphorylation and activation by CRAF, akin to a modeled MEK-KSR2- BRAF regulatory complex (Brennan et al., 2011). Hence, we tested whether ^F129LMEK1 in DDR5, but not DDR4, would be more abundantly associated physically with ^V600EBRAF.

Accordingly, we immunoprecipitated BRAF in the M249 triplet and probed for MEK1 and MEK2 in the immunoprecipitates. Consistently, much more MEK1 and MEK2 were detected in complex with BRAF in ^F129LMEK1 -harboring DDR5 (Figure 5A). We then specifically immunoprecipitated MEK1 and detected a dramatically higher BRAF level bound to MEK1 in DDR5 (Figure 5B). However, the pattern of BRAF-MEK2 binding was reversed; we detected more BRAF bound to MEK2 in DDR4 (Figure 5C). MEK2 in DDR4 was also associated with the highest level of activationassociated phosphorylation at S226, consistent with MEK2 recruitment to and activation by a BRAF-containing complex. Under the same conditions, we were unable to detect CRAF or KSR2 in BRAF, MEK1, or MEK2 immunoprecipitates. These data suggest that the supraphysiologic level of ^V600EBRAF in DDR4 recruits both ^WTMEKI and ^WTM2K2, whereas the ^V600EBRAF level overexpressed to a lesser extent in DDR5 recruits F129L_{M EK1} preferentially over ^WTMEI^. [00102] We also assessed the relative phosphorylation status of MEK1 and MEK2 in DDR4 and DDR5 16 hr after treatment with BRAFi+MEKi versus M249 P treated with DMSO.

Interestingly, we observed that only DDR4, but not DDR5, harbored an enhanced level of activation-associated MEK1 and MEK2 phosphorylation (Figure 5D). In both DDR4 and DDR5, MEK1 displayed increased levels of ERK-dependent negative feedback

phosphorylation on T291 within its proline-rich region of the kinase domain, which is not present on MEK2, suggesting that the time-cumulative ERK activities are far greater in DDR4 and DDR5 despite BRAFi+MEKi treatment than in P M249. DDR5 harbored the highest level of D-MEK1 T291, which has been shown to reduce MEK1-MEK2 heterodimerization and MEK2 S226 phosphorylation (Catalanotti et al., 2009) and may also explain the reduced p- MEK1 S222 level (Figures 5D).

[00103] We then sought to reconstitute ^F129LMEK1-^V600EBRAF interaction and its functional role in DDR. We had observed that the majority of MEK1 and MEK2 mutations thus far detected specifically in melanomas with clinically acquired BRAFi, MEKi, or BRAFi+MEKi resistance (Emery et al., 2009; Shi et al., 2014; Van Allen et al., 2014; Villanueva et al., 2013; Wagle et al., 2011 , 2014) cluster three dimensionally in or proximal to helices A and C (Figure 5E; Movie available in connection with online publication of Moriceau et al., 2015, Cancer Cell 27(2):240-56). Specifically, in M249 P, we minimally overexpressed a series of FLAG-tagged MEK1 constructs and coexpressed either hemagglutinin (HA)-tagged ^WTBRAF or ^V600EBRAF, both at high levels akin to DDR5 (Figures 5F and 12). We then immunoprecipitated protein complexes via FLAG and detected MEK1, BRAF, and HA levels. Importantly, both ^F129LMEK1 and ^Q56PMEK1, which is homolog to ^Q60pMEK2, displayed dramatically enhanced and preferential interaction with overexpressed ^V600EBRAF relative to ^WTMEKl Anti-BRAF signals detected in the FLAG immunoprecipitates presumably contained both endogenous and exogenous ^V600EBRAF. Thus, these data support the notion that BRAFi+MEKi treatment in melanoma selects for MEK1 or MEK2 mutations that impact a discrete structural subdomain and leads to a conformation favoring physical association with overexpressed ^V600EBRAF.

[0104] To assess the functional relevance of a ^V600EBRAF-^MUTMEK complex, we searched for clues of a BRAF-MEK physical interaction interface (Figure 13). Based on prior structural data of MEK1-BRAF (Haling et al., 2014), vemurafenib-bound ^V600EBRAF (Bollag et al., 2010), and MEK1-KSR2 (Brennan et al., 2011) and structural alignments of vemurafenib-bound

V^600EBRAF with BRAF or KSR2, we hypothesized a regulatory ^V600EBRAF-^MUTMEK complex where ^V600EBRAF R662 makes critical contacts with MEK residues in one complex interface (Figures 6A and 6B). We predicted that the R662L substitution in ^V600EBRAF would disrupt this face-to-face ^V600EBRAF-^MUTMEK interaction and attenuate the DDR phenotype. Ectopic expression of vector, HA-^WTBRAF, HA-^V600EBRAF, and HA-^V600E/Ree2LBRAF in ^WTBRAF

HEK293T cells revealed that the R662L substitution did not interfere with the ^V600EBRAF kinase activation status in the absence of MAPKi (Figure 6C). We then engineered M249 P to stably express a FLAG-^F129LMEK1 or FLAG- ^Q60pMEK1 along with HA-tagged WT or various mutant BRAF at levels akin to M249 DDR5 (Figure 6D). After BRAFi+MEKi treatment (1 mM, 16 hr), anti-FLAG immunoprecipitation followed by western blots revealed that both MEK1 mutants most abundantly interacted with ^V600EBRAF, consistent with previous results (Figure 5F). Importantly, the R662L mutation in the context of ^V600EBRAF strongly abolished this enhanced ^V600EBRAF-^MUTMEK1 complex and reduced the overall p-ERK levels.

V600E/R509HBRAF also appeared to display reduced interaction with ^MUTMEK1 but without a reduction in the p-ERK levels, suggesting that this apparent reduction was due to loss of BRAF dimers (Figure 6A) (Haling et al., 2014) or higher-order oligomers (Nan et al., 2013) brought down by anti-FLAG. Consistently, whereas engineered M249 P lines highly overexpressing ^V600EBRAF or minimally overexpressing V600E/R509^HBRAF together with a MEK1 mutant were able to resist robustly BRAFi+MEKi at 1 mM, those cell lines expressing

V600E/R662L_{BRAF or} wr_{BRAF a}|_0ng _{witn an MEK1 mu}tant grew poorly over 28- or 32-day treatments with BRAFi+MEKi (Figure 6E). Taken together, these studies (Figures 4, 5, and 6; Figures 11-13) highlighted a critical role of upstream MAPK reactivation, i.e., upregulation of the ^V600EBRAF-CRAF-MEK complex, in the MAPKi resistance phenotype. Buildup of this plastic complex is dependent on the degree of BRAF and/or MEK inhibition and likely other cell context determinants. In the extreme case of DDR, alternative mechanisms to upregulate this complex can be achieved by ^V600EBRAF (variably overexpressed) interacting with ^CRAF or with MUTMEK.

[0105] Melanoma Cells with Acquired Resistance to BRAFi+MEKi Display Exquisite Dual Drug Addiction

[0106] It has been reported recently that patient-derived xenografts with acquired resistance to BRAFi driven by ^V600EBRAF amplification or RNA overexpression could potentially be counterselected during periods of BRAFi withdrawal (Thakur et al., 2013). We thus tested the degree to which M249 DDR4 and DDR5 were addicted to each (BRAFi or MEKi) or both (BRAFi+MEKi) inhibitors during long-term clonogenic growth. Three days after seeding, DDR4 and DDR5 cells were kept continuously on both inhibitors, washed from both, or replenished with only one of the two inhibitors. Both DDR4 and DDR4 were strongly addicted to continuous treatment with BRAFi+MEKi (Figure 7A). The loss of viability after acute BRAFi+MEKi washout could not be rescued by a dose of ERKi (1 mM) sufficient to strongly suppress the rebound in p-ERK resulting from drug withdrawal (Figure 7B; Figure 14A). Additionally, this "high" dose of ERKi could resensitize DDR4 and DDR5 to either BRAFi or BRAFi+MEKi, consistent with prior short-term MTT results (Figure 3A). Notably, the antigrowth and antisurvival effect of double-drug withdrawal was comparable to that of ERKi alone or ERKi plus BRAFi (Figure 7B). However, ERKi at a suboptimal dose (0.1 mM), which

could suppress the rebound p- ERK levels induced by acute double-drug withdrawal (Figure 14B), completely rescued the antigrowth and antisurvival effects of BRAFi+MEKi withdrawal and partially "erased" the antigrowth and antisurvival effects of single BRAFi or MEKi withdrawal (Figure 7C). Importantly, a suboptimal dose of ERKi could be antigrowth and antisurvival only if DDR4 and DDR5 were continuously treated with BRAFi+MEKi. We then sought evidence consistent with melanoma regression in patients who have been

discontinued on MAPK-targeted therapies due to disease progression or acquired drug resistance. From evaluable patients with melanoma who were treated with BRAFi+MEKi (n = 15) or single-agent BRAFi (n = 16) therapies (Table 4), we retrospectively collated radiologic images before and/or during disease progression and compared them to images, when available or feasible, after a variable time off therapies (Figures 14C and 14D). Although specific clinical examples of tumor regression after cessation of BRAFi+MEKi therapy could be identified, overall disease stabilization or uniform tumor regression leading to clinical remission could not be achieved. Moreover, only cases of tumor growth deceleration could be observed for melanomas after cessation of single-agent BRAFi therapy. Thus, the drug addiction phenotype can be readily elicited in DDR cell lines only if MAPK inhibition was reversed acutely and completely, and additional factors may modulate or mitigate this phenotype clinically.

[0107] Table 4: Clinical characteristics of patients followed for melanoma tumor regression or growth deceleration after cessation of MAPK-targeted therapies.

VICC, Vanderbilt-lngram Cancer Center, USA

UCSF, University of California, San Francisco, USA

UCLA, University of California, Los Angeles, USA

RM, Royal Marsden, London, UK [0108] Given the strong degree of double-drug addiction noted with both DDR4 and DDR5, we asked whether this would be generalizable across different cellular contexts and to melanoma cells with acquired resistance to BRAFi treatment alone. Interestingly, we found that melanoma cell lines adapted to growth with BRAFi+MEKi far more consistently displayed drug addiction (Figure 7D). Also consistent was the observation that melanoma cell lines with DDR displayed a greater rebound in p-ERK levels after drug washout (Figure 7E). This greater rebound was not necessarily due to the maximal p-ERK levels upon withdrawal of drugs but rather due to the very low p-ERK levels in the presence of both drugs (i.e., stronger on-target pathway suppression). Quantification of the fold changes in p-ERK levels (Figure 7E) and in clonogenic growths (Figure 7D) showed that they are strongly negatively correlated. Thus, melanoma cells with DDR displayed a stronger rebound in p-ERK levels and drug addiction upon drug withdrawal, when compared to melanoma cells with single-drug resistance withdrawn from BRAFi (Figure 7F). This p-ERK rebound is indicative of drug addiction since a suboptimal dose of ERKi could rescue cells from double-drug withdrawal- induced loss of fitness (Figure 7C).

[0109] DISCUSSION

[0110] The understanding of how BRAF mutant melanomas frequently acquire BRAFi resistance via several distinct mechanisms, which thematically reactivate the MAPK pathway, has provided foundational rationale to combined BRAF/MEK inhibition to suppress such mechanisms. The ensuing translational effort has led to this combination supplanting BRAFi monotherapy in the clinic. This study of genetic alterations in melanomas with acquired BRAFi+MEKi resistance has provided unexpected insights. First, we detected alterations affecting similar genes known to be responsible for acquired BRAFi resistance, which suggests that the gene dosage or concurrence of these mutations may impart altered molecular interactions promoting BRAFi+MEKi resistance. The exaggerated genetic configurations encompassed gain-of-function (e.g., ^V600EBRAF ultra-amplification, ^G12RNRAS amplification) and LOF (e.g., F127VPTEN, deletions affecting PTEN, CDKN2A, DUSP4) alterations, and their combinations. Second, focusing on MAPK reactivation, we uncovered a highly plastic or tunable RAF-MEK complex resulting from mutations (single-nucleotide variants [SNVs] and/or CNVs). For instance, supraphysiologic levels of ^V600EBRAF

allosterically relay oncogenic MAPK signaling via back-to-back interactions with CRAF.

Moreover, moderately overexpressed levels of ^V600EBRAF likely regulate MEK1 and MEK2 activation via a face-to-face complex. These altered molecular interactions underscore an intrinsic limitation of combined BRAF and MEK inhibition and predict potential limitations of further downstream inhibitors (e.g., ERKi) in overcoming acquired BRAFi+MEKi resistance. [0111] Thus, we have shown how (1) quantitative genetic alterations or gene dosage impact qualitative modesof signaling and (2) combinatorial alterations might be selected to impact survival signaling cooperatively (Figure 8). MEK1 and MEK2 mutants with alterations residing in or proximal to the helices A andC substructures share an increased ability to form an activation-associated complex with ^V600EBRAF, especially when both BRAF and MEK mutants are moderately overexpressed. Moreover, a proposed MUTMEK-^V600EBRAF heterodimer interface strongly suggests that such a face-to-face physical interaction involves

predominantly a kinase-independent or regulatory function of ^V600EBRAF. Together, these data indicate a ^V600EBRAF-CRAF-MEK signaling loop that is highly susceptible to upregulation via single or multiple convergent genetic (and likely nongenetic) alterations.

[0112] Our study of melanoma cell lines with acquired resistance to combined BRAF and MEK inhibition has revealed insights into recent clinical studies. For instance, melanoma cell lines with preexisting BRAFi resistance augment preexisting mechanisms quickly as they adapt to combined BRAF and MEK inhibition. This is consistent with the clinical observation that patients who progressed on BRAFi or MEKi monotherapies infrequently respond to the addition of the other inhibitor, and, for those who do respond sequentially, the responses are generally highly transient. Furthermore, the importance of an MAPKi resistancerelated complex has certain translational implications. Successful strategies targeting this tunable- combinatorial signaling complex may include those inhibiting CRAF function (e.g., omni- or pan-RAF inhibitors), ^V600EBRAF-CRAF interaction, ^V600EBRAF-MUTMEK interaction or scaffolding, and MEK activation (e.g., phosphorylation by RAF). These strategies could be built around continued inhibition of mutant BRAF and MEK or alternating regimens. In our studies, the efficacy of an ERK inhibitor in overcoming acquired BRAFi+MEKi resistance was nuanced and depended on the experimental contexts, e.g., ERKi alone at lower

concentrations promoted survival and growth of BRAFi+MEKi-resistant melanoma cells in both shortand long-term assays.

[0113] While the buildup of a ^V600EBRAF-CRAF-MEK complex ultimately limited the efficacy of combined BRAF and MEK inhibition in melanoma, this signaling complex appeared to be poised to deliver a lethal dose of signaling once both inhibitors were efficiently and acutely removed (Figure 8). Melanoma cells with fully acquired BRAFi+MEKi resistance were much more sensitive to drug withdrawal than those with acquired resistance to BRAFi alone. It is possible that in vivo factors, such as tumor heterogeneity (e.g., subpopulations with reversible drug tolerance but without drug addiction), 3D cell-cell contacts, microenviromental signals, and/or host pharmacokinetic considerations, could render drug addiction a clinically intractable phenotype. In this light, the hypothesis of intermittent therapy with combined BRAFi and MEKi to delay acquired resistance will be tested prospectively within a large

Primers for NRAS cDNA Sanger sequencing

[0138] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

What is claimed is:

1. A method of predicting or detecting the development of acquired resistance to therapeutic effects of combined B-RAF/MEK inhibitor therapy in a patient suffering from cancer, the method comprising:

(a) assaying a sample obtained from the patient for a measure of combined B- RAF/MEK inhibitor therapy resistance, wherein the measure of resistance is selected from:

(13) ^V600EBRAF ultra-amplification;

(14) LOF ^F127VPTEN expression;

(15) homozygous PTEN deletions;

(16) ^G12RNRAS with selective mutant allele amplification;

(17) a homozygous CDKN2A deletion shown in Table 2;

(18) concurrent heterozygous ^Q61KNRAS with homozygous CDKN2A deletion;

(19) ^V600EBRAF amplification concurrent with homozygous CDKN2A deletion;

(20) ^V600EBRAF amplification concurrent with DUSP4 deletion;

(21) ^V600EBRAF amplification concurrent with one or more MEK1 and MEK2

mutations selected from F53Y, Q56P/Q60P, K57N, K59del, V60E, I111S, C121S/C125S, P124IJS G128V, F129L, V154I, E203K, and G276W;

(22) homozygous CDKN2A deletion concurrent with homozygous PTEN deletion;

(23) hemizygous PIK3R1 deletion; and

(24) a genetic profile listed in Table 2 as associated with double drug disease progression (DD-DP);

(b) selecting samples that exhibit a measure of resistance identified in (a); and

(c) identifying a patient whose sample was selected in (b) as susceptible to developing resistance to combined B-RAF/MEK inhibitor therapy.

2. The method of claim 1 , wherein the combined B-RAF/MEK inhibitor therapy comprises treatment with a B-RAF inhibitor selected from vemurafenib and dabrafenib.

3. The method of claim 1 , wherein the combined B-RAF/MEK inhibitor therapy comprises treatment with a MEK inhibitor selected from trametinib, selumetinib, cobimetinib, and binimetinib.

4. The method of claim 1 , wherein the sample is selected from tissue, bodily fluid, blood, tumor biopsy, spinal fluid, and needle aspirate.

5. The method of claim 1 , which is performed prior to treatment with combined B- RAF/MEK inhibitor therapy.

6. The method of claim 1 , which is performed after treatment with combined B-RAF/MEK inhibitor therapy.

7. The method of claim 1 , which is performed during disease progression or clinical relapse on combined B-RAF/MEK inhibitor therapy.

8. The method of claim 1 , which is performed after suspension of combined B-RAF/MEK inhibitor therapy.

9. The method of claim 1 , wherein the cancer is melanoma.

10. The method of claim 1 , further comprising identifying in a patient who is susceptible to developing or has developed resistance to combined B-RAF/MEK inhibitor therapy the form(s) of resistance and method(s) to counter such resistance.

11. The method of claim 1 , further comprising treating the patient with intermittent dosing of combined B-RAF and MEK inhibitors.

12. The method of claim 1 , wherein the assaying comprises targeted sequencing, realtime RT-PCR, Sanger sequencing and/or whole exome sequencing.

13. The method of claim 1 , wherein the assaying comprises contacting the sample with one or more primers selected from SEQ ID NO: 1-106.

14. The method of claim 1 , wherein the ^V600EBRAF ultra-amplification is identified by detecting more than 70 copies of ^V600EBRAF.

15. A method of preventing or suppressing acquired resistance to combined B-RAF/MEK inhibitor therapy in a patient, the method comprising administering to the patient a therapeutic agent selected from the group consisting of:

(a) a suppressor of C-RAF kinase activity;

(b) an inhibitor of activation of MEK1 or MEK2 by C-RAF; and

(c) an inhibitor of activation of ERK1 or ERK2 by MEK1 and MEK2.

16. The method of claim 15, wherein the therapeutic agent is selected from the group consisting of: Omni-RAF, CRAF and RAF paradox breakers, and ERK inhibitors.

17. The method of claim 16, wherein the therapeutic agent is CCT196969, CCT241161 , PLX3397, PLX7904 and/or SCH442984.