WO2013155077A1 - Response markers for src inhibitor therapies - Google Patents

Abstract

Methods for determining whether a subject having cancer is a good candidate for a Src inhibitor therapy. For example, methods of the embodiments can comprise testing a sample from a subject to determine whether cancer cells from the subject have reduced BRAF activity and/or express a mutant BRAF protein. Methods for treating a subject having cancer with reduced BRAF kinase activity and/or that expresses a mutant BRAF protein by administering a Src inhibitor therapy are also provided.

Description

DESCRIPTION RESPONSE MARKERS FOR SRC INHIBITOR THERAPIES

[0001] The present application claims the priority benefit of United States provisional application number 61/621,747, filed April 9, 2012, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The invention was made with government support under Grant No. P50CA70907 awarded by the National Institutes of Health through the National Cancer Institute. The government has certain rights in the invention.

1. Field of the Invention

[0003] The present invention relates generally to the field of molecular biology and oncology. More particularly, it concerns methods for treating cancer and methods for predicting a response to anti-cancer therapies.

2. Description of Related Art

[0004] Lung cancer is the leading cause of cancer-related deaths worldwide. Researchers recently identified two molecular subpopulations of non-small cell lung cancer (NSCLC)— populations with epidermal growth factor receptor (EGFR) mutations and those with EML4-ALK translocations— for which treatment with targeted agents has produced profound clinical responses. This development has elicited a paradigm shift in treating lung cancer: the tumors' genetic characteristics dictate therapy. Nevertheless, definition of clinically relevant genetic aberrations is lacking in about 85% of NSCLC cases.

SUMMARY OF THE INVENTION

[0005] In a first embodiment there is provided a method for identifying a candidate for a Src inhibitor therapy comprising (a) testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein; and (b) identifying the subject as a good candidate for a Src inhibitor therapy if the sample from the subject indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity; or identifying the subject as a poor candidate for a Src inhibitor therapy if the sample from the subject does not indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity.

[0006] In a further embodiment there is provided a method for identifying a good candidate for a Src inhibitor therapy comprising testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein, thereby identifying the subject as a good candidate for a Src inhibitor therapy.

[0007] In some aspects, testing the sample in accordance with the embodiments comprises measuring BRAF kinase activity in the sample. In certain aspects, testing a sample comprises determining whether cells of the cancer comprises a BRAF gene encoding a protein with reduced kinase activity. For example, the BRAF gene can encode a protein comprising an amino acid substitution or deletion at position Y472 (e.g., a Y472C amino acid substitution). Thus, in some aspects, testing a sample comprises determining all or part of a BRAF gene sequence in the sample. In some aspects, a sample is a blood sample, a tissue sample, a lymph sample or a saliva sample.

[0008] Certain aspects of the embodiments concern identifying a subject that is a good candidate for a Src inhibitor therapy, such as a subject that will have a favorable anticancer response to a Src inhibitor therapy. For example, a good candidate for a Src inhibitor therapy can be, without limitation, a subject that is predicted to have reduced in tumor size or burden, reduced tumor growth, reduced tumor-associated pain, reduced cancer associated pathology, reduced cancer associated symptoms, cancer non-progression, cancer remission, reduced cancer metastasis, or increased survival time in response to a Src inhibitor therapy. In further aspects, a method of the embodiments comprises providing a report, such as a written or electronic report, indicating whether the subject is a good candidate or a poor candidate for a Src inhibitor therapy. In some cases, such a report is transmitted to a third party such as a hospital, a doctor, an insurance company or a healthcare provider.

[0009] In a further embodiment, there is provided a method for treating a subject having a cancer, wherein it was determined that cancer cells from the subject comprise reduced BRAF kinase activity relative to a control level, the method comprising administering a Src inhibitor therapy (e.g., a Src kinase inhibitor) to the subject. For example, a method can comprise administering a Src inhibitor therapy 1, 2, 3, 4, 5, 6 or more times to the subject over a period of days, weeks or months. In further aspects, a method of the embodiments further comprises administering at least a second anti-cancer therapy to the subject, such as chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti- angiogenic therapy or cytokine therapy. Thus, in some aspects, a composition is provided for the treatment of a subject having cancer comprising cancer cells with reduced BRAF kinase activity relative to a control level, the composition comprising a therapeutically effective amount of a Src inhibitor.

[0010] In yet a further embodiment there is provided a method for treating a subject having a cancer comprising administering to the subject an effective amount of a Src inhibitor in conjunction with (e.g., before, after or concurrently with) a RAF inhibitor. For example, the Src inhibitor can be a Src kinase inhibitor, such as bosutinib, saracatinib, danusertib, VP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib. In certain aspects, the RAF inhibitor is a BRAF inhibitor, such as a BRAF specific inhibitor. Examples of RAF inhibitors include, without limitation, vemurafenib, GSK2118436 and sorafenib. In further aspects, a composition is provided for the treatment of a subject having cancer comprising a therapeutically effective amount of a Src inhibitor and a RAF inhibitor.

[0011] In certain aspects a subject of the embodiments is a subject having or diagnosed with a cancer, such as a lung, breast, brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck, esophageal, liver, skin, kidney, leukemia, bone, testicular, colon, or bladder cancer. For example, the cancer can be a lung cancer such as a non-small cell or a metastatic lung cancer.

[0012] In certain aspects, a Src inhibitor therapy of the embodiments is a Src kinase inhibitor. For example, the Src kinase inhibitor can be bosutinib, saracatinib, danusertib, VP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib. [0013] In still a further embodiment there is provided computer-readable medium that, when executed by a computer, causes the computer to perform operations comprising (a) receiving information corresponding to a BRAF kinase activity in a sample from a subject having a cancer; and (b) determining a relative level of BRAF kinase activity compared to a reference level, wherein reduced BRAF kinase activity relative to the reference level indicates the subject is a good candidate for a Src inhibitor therapy. For example, the information corresponding to a BRAF kinase activity can comprises all or part of a BRAF gene sequence. Thus, in some aspects, determining a relative level of BRAF kinase activity comprises determining a change in all or part of a BRAF gene sequence compared to a reference sequence, wherein a change in the BRAF gene sequence corresponding to reduced BRAF activity indicates that the subject is a good candidate for a Src inhibitor therapy. For example, a reference level of the embodiments can be all or part of a wild type BRAF gene sequence or all or part of a BRAF gene sequence corresponding to protein with increased or decreased kinase activity relative to a wild type sequence. In still further aspects, receiving information in accordance with the embodiments comprises receiving from a tangible data storage device information corresponding to a level of BRAF kinase activity in a sample from a subject having a cancer.

[0014] In further aspects a tangible computer-readable medium of the embodiments further comprises a computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the relative level of BRAF kinase activity to a tangible data storage device. In yet further aspects, a tangible computer-readable medium comprises a computer-readable code that, when executed by a computer, causes the computer to perform operations further comprising (c) calculating a diagnostic score for the sample, wherein the diagnostic score is indicative of the probability that the subject will respond to a Src inhibitor therapy (e.g., whether the subject is a good candidate for a Src inhibitor therapy). [0015] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0016] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0017] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0018] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0020] FIG. 1. ^Y472CBRAF is biochemically similar to ^G466VBRAF but different from 6^00EBRAF. A) Kinase activity in Flag-tagged BRAF proteins with the noted mutations. After immunoprecipitation (IP) from COS7 cells and incubation with kinase-dead MEK (substrate) and ATP, MEK phosphorylation was measured using Western blotting and quantitated with ImageJ. (B-D) The effect of the expression of mutated BRAF proteins on MEK and ERK activity in intact cells. Western blotting was performed in (B) COS7, (C) H661, and (D) H226 cells transfected with BRAF constructs. E) The activity of CRAF in H661 cells transfected with mutated BRAF, as measured by incubating immunoprecipitated CRAF with kinase-dead MEK (substrate) and ATP. F) CRAF kinase activity and BRAF-CRAF binding, as measured in COS7 cells transfected with FLAG-tagged BRAF with the indicated mutations by kinase activity (IVKA) or IP of CRAF followed by blotting with anti-Flag and anti-BRAF antibodies (binding). WT, wild-type.

[0021] FIG. 2. NSCLC cells with an inactivating BRAF mutation undergo senescence when exposed to dasatinib. A-D) Biological effects of dasatinib in NSCLC cells with BRAF mutations. NSCLC cells were incubated with 150 nM dasatinib or a control vehicle for 72 hours followed by β-galactosidase (A) staining and (B) quantification, (C) cell cycle analysis using propidium iodide staining and FACS analysis, or (D) BrdU incorporation. E) The effect of dasatinib on downstream signaling as measured by Western blotting of NSCLC cells incubated with 150nM dasatinib for 24 hours. (F) The reversibility of dasatinib-induced senescence. Call2T cells were incubated with 150nM dasatinib or fresh medium (vehicle, but no drug) for the indicated times (left panel) and senescence was measured with β-galactosidase staining using light microscopy. *P <0.05. **P <0.001. [0022] FIG. 3. Expression of kinase inactive and active BRAF leads to sensitivity and resistance to dasatinib, respectively, in NSCLC cells. (A, B, D) Cell viability was assayed following dasatinib treatment in NSCLC cells transfected with BRAF constructs. Following transfection with the noted BRAF constructs (A) H661, (B) H226, and (D) HI 666 cells were and incubated with dasatinib at increasing doses for 72 hours and cell viability was estimated using the MTT assay. The half-maximal inhibitory concentration (IC₅₀) of dasatinib was calculated for H661. C) Apoptosis in H661 cells expressing BRAF with the noted mutations as measured using the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling assay. [0023] FIG. 4. Dasatinib indirectly inhibits CRAF activity in cells with an inactivating BRAF mutation but not in cells with ^BRAF or ^600EBRAF. A) Kinase activity of isolated BRAF and CRAF incubated with dasatinib. Purified recombinant BRAF and CRAF were incubated with 100 nM dasatinib in the presence of inactive MEK-1 and ATP. Kinase activity was measured by phosphorylation of MEK-1. B) The effect of dasatinib on the kinase activity of CRAF in intact cells expressing various BRAF constructs. COS-7 cells were transfected with Flag-tagged CRAF along with HA-tagged BRAF constructs with the noted mutations 24 hours prior to dasatinib treatment. Transfected cells were incubated with 150 nM dasatinib or vehicle for 24 hours followed by immunoprecitation with an anti-Flag antibody. The resulting lysate was subjected to a Western blot with anti-Flag and anti-HA antibodies (lower two rows) and CRAF kinase activity was measured by incubating Flag- immunoprecipitated CRAF with kinase-dead MEK-1 (substrate) and ATP (IVKA, top row). MEK activation was quantitated and normalized with total FLAG. (C) The effect of CRAF knock down (KD) on NSCLC cell number in cells with endogenous BRAF mutations. Untransfected Catl2T, H1666, H322, and H661 cells were incubated with CRAF siRNA, and their viability was estimated using an MTT assay. In all cases, Western blotting was used to confirm CRAF knockdown. At 120h Call2T cells lost dasatinib sensitivity and CRAF expression recovered.

[0024] FIG. 5. Dastinib induces RAF dimerization in NSCLC cells which is necessary for sensitivity in cells expressing kinase impaired BRAF. (A) Dasatinib induced RAF dimerization. NSCLC cells were incubated with 150 nM dasatinib for 72 hours then CRAF was immunoprecipitated. The resulting lysate was split with part used for blotting with antibodies to BRAF or CRAF (to measure dimerization) and part used to measure CRAF kinase activity using MEK as a substrate. (B-C) Introduction of a mutation that interferes with RAF dimerization abrogates the effects of kinase deficient BRAF on dasatinib sensitivity. H661 cells transfected with the BRAF constructs as indicated were subjected to Western blotting (B) and an MTT assay (C). [0025] FIG. 6. Dasatinib enhances the cytotoxicity of PLX4032 in cancer cells resistant to dasatinib. Dasatinib-resistant NSCLC cells were incubated with single agent dasatinib (50-600 nM) or single agent PLX5032 (1000- 12000 nM) or in combination of both (dasatinib:PLX4032 = 1 :20) for 72 h, and their viability was estimated using an MTT assay.

[0026] FIG. 7. (A) Radiographic images of PX's primary lung tumor and paraspinal metastasis, demonstrating that only a residual lung nodule remains over 3 years after the completion of treatment with dasatinib alone for 12 weeks. (B) Kaplan-Meier overall survival and progression free survival curves for the 34 patients enrolled on the dasatinib phase 2 study at a median of 36 months of follow up.

[0027] FIG. 8. Alignment of the human BRAF (Homo; ; SEQ ID NO:3) amino acid sequence with that of mice (Mus; SEQ ID NO: L>, rats (Rattus; SEQ ID NO:2), chickens (Gallus; SEQ ID NO:4), and frogs (Xenopus; ; SEQ ID NO:5). The tyrosine residue marked in red represents the Y472 site in humans, which is conserved in all tested evolutionary distant organisms.

[0028] FIG. 9. NSCLC cells with kinase inactive BRAF mutations did not undergo apoptosis when exposed to 150nM dasatinib for 72 hours as measured by TUNEL staining.

[0029] FIG. 10. Cells with inactive BRAF undergo senescence in the presence of dasatinib. Immunofluorescence microscopy was done with Call2T, HI 666 (A), H661 and H2987 (B) cell lines treated with vehicle or 150 nM dasatinib for 72 hours. Cells were labeled with senescence-associated heterochromatin foci marker HPl-γ and counterstained with DAPI. Induction of HPl-γ in Dasatinib treated cells is significantly higher in Call2-T and HI 666. (C) Fold change in HPl-γ expressing cells in dasatinib treated group compared to vehicle control in three independent experiments.

[0030] FIG. 11. HI 666 cells were incubated with 100 nM dasatinib for the indicated times followed by replacement with fresh media (schedule per left panel) and senescence was quantitated with β-galactosidase staining using light microscopy. (*P value <0.05). [0031] FIG. 12. H661 cells transfected with inactivating BRAF mutations show increased sensitivity to dasatinib. H661 cells were transfected with the noted BRAF constructs and incubated with increasing doses of dasatinib for 72 hours. Their viability was estimated using an MTT assay. These data were used to generate the data presented in FIG. 3A.

[0032] FIG. 13. Transfection with mutant or wt BRAF does not affect cell number. HI 666 and H661 cells were transfected with the noted BRAF constructs and cell number was estimated 72 hours later using the MTT assay.

[0033] FIG. 14. NSCLC cells with an inactivating BRAF mutation are sensitive to CRAF knockdown (KD). H661 cells expressing the noted BRAF proteins were incubated with CRAF siRNA, and their viability was estimated using an MTT assay. In all cases, Western blotting was used to confirm CRAF knockdown.

[0034] FIG. 15. Kinase inhibitor- induced RAF dimerization does not result in drug sensitivity or senescence. NSCLC cells were incubated with the noted drugs (dasatinib: 150 nM, nilotinib: 2 μΜ, bosutinib: 1.5 μΜ and AZD0530: 5 μΜ respectively) for 72 hours followed by immuoprecipitation with CRAF from Call2T cells (A), MTT assay (B), or β- galactosidase staining (C).

[0035] FIG. 16. Dasatinib does not have any BRAF mutation-specific changes in BAD or JNK phosphorylation. The noted NSCLC cell lines were incubated with 150 nM dasatinib for 72 hours followed by Western blotting with the noted antibodies.

[0036] FIG. 17. Inhibition of c-Src does not affect cell viability in NSCLC harboring a kinase inactive BRAF mutation. H1666 cells were incubated with AZD0530, c-Src siRNA, or controls. (A and B) Western blotting confirmed c-Src knock down or inhibition . (C) Neither c-Src siRNA nor controls affect cell viability 72 hours following transfection. (note: The MTT assay for AZD0530 is included in FIG. 15B).

[0037] FIG. 18. Dasatinib enhances the cytotoxicity of sorafenib in cancer cells resistant to dasatinib. Dasatinib-resistant head and neck cancer (top four panels) and NSCLC (bottom four panels) cells were incubated with 100 nM dasatinib and 1-10 μΜ sorafenib for 72 hours, and their viability was estimated using an MTT assay. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0038] Metastatic NSCLC is a common and fatal disease with a 4-year survival rate of only 2%, but personalized therapies targeting specific genetic aberrations in NSCLC tumors are remarkably successful. A patient was identified with stage IV NSCLC with long term disease control by single-agent dasatinib therapy alone. His tumor harbored a kinase inactivating ^Y472CBRAF mutation that was likely responsible for its unusual sensitivity to dasatinib. Although effective treatments are available for melanoma patients with activating BRAF mutations, no such therapies are available for cancer patients who harbor inactivating mutations. [0039] No inactivating BRAF mutations were identified in any other patients in the clinical trial, all of whom were nonresponders. As with previously characterized kinase inactivating BRAF mutations, ^Y472CBRAF expression led to CRAF, MEK, and ERK activation. Also, NSCLC cell lines with endogenous inactivating BRAF mutation underwent senescence when exposed to dasatinib; the cell lines' dasatinib sensitivity reversed with the overexpression of active BRAF. Whereas NSCLC cells transfected with an activating BRAF mutation were more resistant to dasatinib than were controls, transfection of NSCLC cells with kinase impaired BRAF led to their increased dasatinib sensitivity. These data and PX's pattern of clinical response are consistent with the conclusion that his tumor underwent senescence when exposed to dasatinib owing to its inactivating BRAF mutation. The mechanism of dasatinib-induced senescence and apoptosis in NSCLC cells expressing kinase impaired BRAF is unknown but may relate to increased RAF dimerization leading to ERK activation consistent with the paradigm of oncogene-induced senescence that occurs following moderate BRAF or KRAS activation (1 1). However, the modest effects on ERK suggest that ERK-independent pathways are involved as well. [0040] BRAF mutations occur in only about 4% of NSCLC cases but are more common in other tumors, such as melanoma (50%) and papillary thyroid cancer (40%) (19,20). The majority of BRAF mutations cause activation of the kinase (20). In NSCLC, 57% of mutations are remaining 43% are a mixture of kinase inactivating, activating, and uncharacterized mutations (19). CRAF mutations are rare in all cancers (21). [0041] Interactions between CRAF and BRAF are complex and incompletely understood despite several recent elegant studies in melanoma (4, 5, 22, 23). The pathway is not linear and is further complicated by the multiplicity of signaling molecule components; BRAF and CRAF are similar to each other but do not function identically and are not interchangeable and feedback pathways lead to inactivation of upstream components of the pathway (i.e., a negative feedback amplifier) (24). Additionally, MEK is not the only RAF substrate (25).

[0042] The complexity of BRAF-CRAF interactions is epitomized by the RAF inhibitor paradox. Specifically, inhibition of BRAF activity in cells that express ^600EBRAF results in expected inhibition of ERK activity and subsequent apoptosis; melanomas with 6^00EBRAF respond clinically to BRAF inhibition (26). Paradoxically, BRAF inhibition in cells with active RAS leads to ERK activation via activation of CRAF (4). These observations are supported by previous research demonstrating that CRAF and BRAF heterodimers are more active than is either the CRAF or BRAF homodimer even when the BRAF protomer has an inactivating mutation (23).

[0043] These complex interactions of RAS, BRAF, and CRAF explain the existence and function of inactivating BRAF mutations in cancer cells, which superficially seem to be contrary to natural selection. Activation of CRAF not only allows cells expressing kinase impaired BRAF to survive but also may further promote the cancer phenotype by activating non-ERK-dependent pathways or promoting aneuploidy (25, 27). CRAF activation may also be responsible for BRAF-inhibitor-induced squamous cell carcinomas (26). [0044] Although much of the research involving BRAF has focused on melanoma, the BRAF pathway undoubtedly is active and important in NSCLC. RAS is commonly activated in NSCLC via upstream growth factor receptors or activating mutations. Active KRAS signals predominantly through CRAF, not BRAF (28). The role of CRAF in NSCLC has yet to be elucidated, although CRAF is overexpressed in NSCLC cells, and its forced overexpression leads to lung adenomas in a transgenic mouse model (29).

[0045] Oncogene-induced senescence occurs after activation of oncogenes such as RAS and RAF. Classically, it is mediated by activation of the ρΙό^^/ΕΗ) and/or pi 4^ARF/p53 tumor suppressor pathway. Senescence also can be induced and mediated by other pathways, such as those involving c-Src, STAT3, c-Myc, FOX04, Chk2, and c-Jun-N-terminal-kinase. In the present study, dasatinib induced senescence in HI 666 and Call2T cells but induced apoptosis in H661 cells into which were transfected kinase impaired BRAF mutations. Possible reasons for this discrepancy are that the absolute level of endogenous ^w BRAF, the level of mutant BRAF, or the w mutant BRAF ratio may influence the outcome of dasatinib- based treatment. The level of mutant BRAF expression was higher in the transfected cells, which also harbored a full complement of endogenous "'BRAF, than in non-trans fected cells. [0046] Occasionally, spontaneous tumor regression occurs in melanoma and renal cell carcinoma cases and is thought to be immune mediated. Also, the activating ^V600EBRAF mutation in melanoma may induce an immune response. Although such a possibility cannot be excluded in the case of PX, spontaneous tumor regression is very rare in NSCLC cases. Instead, the dasatinib sensitivity of NSCLC cell lines with kinase impaired BRAF mutations is consistent with the patient having experienced a direct antitumor effect of dasatinib rather than an immune-mediated mechanism.

[0047] Other potential mechanisms not fully explored in these studies are the roles of other dasatinib targets in mediating dasatinib sensitivity. Although no protein expression has been linked to dasatinib sensitivity, Sos et al. demonstrated that copy-number gains influence sensitivity of NSCLC cells to kinase inhibitors (7). H322 cells, which have c-Src amplification, are sensitive to dasatinib. PX's tumor had copy -number gains in several dasatinib targets. Because live tumor tissue specimens are not available from PX, determining whether his tumor was dependent upon HCK, DDR1, EPHA3, or ARG for survival is impossible (30). [0048] In conclusion, it was demonstrated that the kinase inactive mutation

^Y472CBRAF functions similarly to other known inactivating BRAF mutations and was likely responsible for PX's response to dasatinib. Moreover, the potential for synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options. Given that dasatinib and BRAF inhibitors are in clinical use, this work has potential for direct clinical applications.

I. Examples

[0049] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Patient's Tumor Analysis [0050] In a Phase 2 study of dasatinib in 34 patients with systemic therapy-naive stage IV NSCLC the sole responder was a male former smoker (PX) who had a profound, durable response (2). Over the 12 weeks of dasatinib-based therapy, PX had a partial response as assessed by both tumor size and metabolic activity and his metastatic tumor (in paraspinal muscle) continued to shrink after therapy was stopped. At the end of therapy, the diameter of the metastasis was 2.8 cm, with a standardized uptake value (SUV) of 17. At 17 weeks, accurately measuring the metastasis on a computed tomography (CT) scan was difficult, but the SUV was 1 1. At 21 weeks, the SUV was 4.5. At 32 weeks, the mass was undetectable on CT and positron emission tomography scans (2). Subsequent follow up shows that PX remains free of active cancer 4 years after the initial diagnosis and has not received any other cancer therapy. PX still has a 2-cm lung nodule that has no detectable metabolic activity on PET and that has been stable on CT scans for 4 years (FIG. 7A). The median progression free survival was 1.4 months and the median overall survival was 15.6 months (FIG. 7B). Additional studies of PX's tumor tissue were performed to identify the underlying mechanism of dasatinib sensitivity. [0051] PX's tumor did not harbor any EGFR or KRAS mutations by intron-based polymerase chain reaction (PCR) οΐΚ-Ras exons 1 and 2 (codons 12, 13, and 61) and EGFR exons 18-21 as previously published (2). Likewise, no ALK gene rearrangements were detected by fluorescence in situ hybridization; no c-Src mutations were detected by intron- based PCR of exons 7-10; nor were any discoidin domain receptor 2 (DDR2) mutations detected using conventional Sanger Sequencing of all exons as previously described (6). Immunohistochemistry revealed that the tumor did express total and phosphorylated EphA2, c-Kit, and PDGFRa (Table 1). PX did not harbor any germ line BRAF or KRAS mutations by intron-based PCR of BRAF exons 1 1 and 15 and K-Ras exons 1 and 2 (codons 12, 13, and 61) of DNA isolated from his peripheral blood lymphocytes. [0052] To identify novel mutations or changes in gene copy number in PX's tumor, the MassARRAY system (Sequenom) was used and aCGH performed. No mutations were detected among the 40 genes tested (Table 2). Using aCGH, several regions of increased and decreased copy numbers were detected (Table 3). Increased copy numbers of the known direct dasatinib targets HCK, DDR1, EPHA3, and ARG (ABL2) were also detected. No copy number changes for LYN, FGR, FYN, SRC, DDR2, EPHB1, EPHB2, EPHB3, EPHA1, EPHA2, EPHA4, TNK2, PTK6, GAK, KIT, PDGFR, KRAS, EGFR, or BRAF were detected.

[0053] Table 1: Immunchistochemistry scores for PX's tumor.

[0054] Table 2: Genes analyzed using mass spectroscopy single-nucleotide polymorphism analysis.

Amino acid Nucleic acid change

position tested

Amino acid Nucleic acid change position tested

Amino acid Nucleic acid change position tested

Amino acid Nucleic acid change position tested

Amino acid Nucleic acid change position tested

Amino acid Nucleic acid change position tested

Amino acid Nucleic acid change position tested

[0055] Table 3: Copy number variation for genes associated with dasatinib targeting and/or NSCLC.

* Dasatinib target-associated gene.

[0056] Table 4: Mutational statuses of patients with NSCLC treated with dasatinib.

RECIST, Response Evaluation Criteria in Solid Tumors; AC, adenocarcinoma; PD, progressive disease; NE, not evalauted; SD, stable disease; PR, partial response; SCC, squamous cell carcinoma.

Example 2 - Identification of a Novel Inactivating Mutation in BRAF

[0057] Because the Sequenom MassARRAY technology is limited in that can only identify candidate mutations in which assays are specifically designed and given the known role of BRAF in oncogene-induced senescence, exons 1 1 and 15 of the BRAF gene were sequenced. These two exons possess many known mutations not included in the panel. The identified mutation ^Y472CBRAF, has not been described previously and occurs in a highly conserved region of exon 1 1 (FIG. 8). The BRAF gene was also analyzed in 19 patients from the original clinical trial for whom DNA adequate for analysis was available and no other inactivating mutations were found (Table 4). [0058] To determine the functional significance of ^Y472CBRAF, site-directed mutagenesis was used to create ^Y472CBRAF, ^G466 BRAF (kinase-impaired), and ^600EBRAF (constitutively active) in a Flag-tagged ^BRAF construct. The constructs were transfected into COS7 cells, the Flag-tagged proteins isolated and tested for kinase activity. As expected, ^V600EBRAF had increased kinase activity and ^G466VBRAF had reduced kinase activity. ^Y472CBRAF showed severely reduced kinase activity that was less than 10% that of ^BRAF (FIG. 1A).

Example 3 - Lung Cancer Cells Expressing ^Y472CBRAF Express Activated MEK, ERK, and CRAF

[0059] Cancer cells that express kinase impaired BRAF mutations still activate MEK and ERK via trans activation of CRAF (4, 5). To determine whether ^Y472CBRAF functions similarly, COS7, H226, and H661 cells (all with ^BRAF) were transfected with the BRAF construct panel and their ERK and MEK activity measured (FIG. IB-ID). As expected, transfection with ^600EBRAF resulted in phosphorylation of ERK and MEK to levels markedly above those in cells transfected with ^wtBRAF. ^Y472CBRAF and ^G466VBRAF both activated MEK and ERK to levels at or above those observed after transfection with ^BRAF. Cells transfected with ^BRAF had similar levels of total BRAF compared to cells transfected with mutant BRAF (FIG. 1C and ID).

[0060] To determine whether ^Y472CBRAF transactivates CRAF, CRAF kinase activity was assessed in H661 cells transfected with the BRAF constructs panel. Cells expressing

G466V_{BRAF or} Y472C_{BRAF CRAp actiyity man d}j_{d mQs e ceUs express}i_ng ^BRAF or (FIG. i ImE). τ L ;ik1 ewise, c _eilils express ;ing G466V Bn>nRATF? o„„r Y ^Y4⁴7^/2C^L _TBRAF had higher BRAF-CRAF binding than did cells expressing BRAF or ^VWJU¾RAF (FIG. IF). These results confirmed that ^Y472CBRAF functions in the RAS/RAF/MEK/ERK pathway similarly to previously characterized BRAF proteins with inactivating mutations.

Example 4 - Dasatinib Leads to Senescence and Apoptosis in Lung Cancer Cells

Expressing Kinase impaired BRAF

[0061] To determine whether ^Y472CBRAF was related to PX's response to dasatinib, w a panel of NSCLC cell lines was tested for their sensitivity to dasatinib. After extensive searching in multiple databases, only two NSCLC cell lines were identified with known inactivating BRAF mutations: H1666 and Call2T. These two lines, along with one cell line expressing ^BRAF (H322), were sensitive to dasatinib (Table 5). Researchers previously characterized H322 cells as having c-Src amplification, which drives their sensitivity to dasatinib (7). All other lines tested were resistant to dasatinib, including those with activating BRAF mutations (Table 5).

[0062] Significant apoptosis was not observed when H1666 or Call2T cells were treated with dasatinib (FIG. 9), but the cells did undergo cell-cycle arrest with an increased proportion of cells in Gl, decreased proliferation with reduced BrdU incorporation, and they stained for β-galactosidase and HPI-gamma, indicating senescence (FIGs. 2A-2D and 10). Consistent with this finding, dasatinib reduced phosphorylated Rb in HI 666 and Call2T (FIG. 2E). [0063] To determine whether dasatinib-induced senescence was reversible, Call2T and HI 666 cells were incubated with dasatinib for 6-96 h followed by drug removal and measurement of β-galactosidase expression (FIGs. 2F, 1 1). Induction of significant tumor cell senescence required 72 h of exposure to dasatinib and was largely irreversible by 96h.

[0064] To further study the effects of inactivating BRAF mutations on dasatinib sensitivity, H661 and H226 cells were transfected with the panel of BRAF constructs. Expression of the inactivating mutations increased the cells' sensitivity to dasatinib, whereas expression of ^600EBRAF induced further resistance (FIGs. 3A, 3B, and 12). In contrast HI 666 and Call2T cells, which have endogenous BRAF inactivation (^G466 BRAF), H661 cells, transfected with kinase impaired BRAF, underwent apoptosis and not senescence when exposed to dasatinib (FIG. 3C). [0065] To confirm the importance of the inactivating BRAF mutations in mediating dasatinib sensitivity in HI 666 cells, these cells were transfected with kinase active BRAF (i.e., ^600EBRAF or ^BRAF) and treated them with dasatinib (FIG. 3D). Overexpression of kinase active BRAF increased resistance to dasatinib, confirming the role of kinase inactive BRAF in mediating dasatinib sensitivity. None of the constructs in the panel of BRAF constructs had a significant effect on cell number in untreated cells (FIG. 13).

[0066] Table 5: Lung cancer cell sensitivity to dasatinib

BRAF Kinase

mutation Dasatinib activity

Cell line BRAF type IC₅₀ (nM) (fold wt)* Other known mutations or polymorphisms

H1666 G466V Inactivating 158 0-65 CDKN2A, STKl 1

H322 wt wt 142 1 TP53, CDKN2A, STKl 1

Cal-12T G466V Inactivating 238 0-65 TP53, CDKN2A

H661 wt wt 1484 1 TP53, CDKN2A, SMARCA4

H460 wt wt >2500 1 KRAS, CDKN2A, STKl 1, PIK3CA

H1568 wt wt 3321 1 None known

H226 wt wt >5000 1 TP53

A549 wt wt >5000 1 KRAS, CDKN2A, SMARCA4, STKl 1

H522 wt wt >5000 1 TP53

H2087 L597V Activating >5000 64 NRAS, TP53, DGKB, DGKB, PTPRT, ATM, BAI3, HECWl,

HEPH, NLRP4, NTRK3, PDZRN4, PTPRM

H1755 G469A Activating >5000 266 KRAS, TP53

H1395 G469A Activating >5000 266 STKl 1, NLRPl, RTPRT, ATM, BIRC6, NLRP4, TRIM36

HCC364 V600E Activating >5000 478 None known

H2405 In- frame Unknown >8000 ND TP53

deletion

*Based on published values determined using an in vitro kinase assay (33, 34). ND, not done.

Example 5 - Dasatinib Indirectly Inhibits CRAF

[0067] The mechanism by which dasatinib induces senescence in NSCLC cells with kinase-deficient BRAF is unknown. It was hypothesized that dasatinib induces senescence by affecting CRAF function (8). Although it was found that dasatinib did not directly affect CRAF or BRAF kinase activity at relevant concentrations (FIG. 4A), which is consistent with published studies (7, 9), it led to decreased CRAF activity in intact cells that express kinase inactive BRAF (FIG. 4B). To establish the importance of CRAF in NSCLC cells with kinase impaired BRAF, CRAF expression was knocked-down using siRNA in NSCLC cells with kinase active or inactive BRAF. CRAF knockdown in H1666 (^G466VBRAF) and Call2T (^GA66 BRAF), but not H322 (^wtBRAF) or H661 (^wtBRAF) cells affected their viability as estimated using an MTT assay (FIG. 4C), although the effects on Call2T cells were modest. Given the limited number of cells lines expressing endogenous mutated BRAF, a less physiologic approach was used and H661 cells were transfected with kinase active or inactive BRAF. Only cells expressing kinase-deficient BRAF showed reduced cell number after treatment with CRAF siRNA (FIG. 6).

Example 6 - Dasatinib Induces RAF Dimerization

[0068] Consistent with the recent findings of Packer et. al. it was found that dasatinib induced RAF dimerization in cells with a KRAS mutation (A549) (10). In addition, dasatinib induced RAF dimerization in NSCLC cells with kinase impaired BRAF (FIG. 5A). Although dasatinib caused a modest inhibition of RAF kinase activity, when corrected for the increase in total RAF dimers, there was no net inhibition of RAF in Call2T and H1666 cells. Similarly, ERK was activated and p21 expression induced after dasatinib treatment in cells with kinase impaired BRAF consistent with oncogene induced senescence that is observed with KRAS or BRAF activation (FIG. 2E) (1 1). ERK and MEK were inhibited in H661 and A549 cells, which is consistent with their lack of senescence following dasatinib exposure.

[0069] To further investigate the role of BRAF/CRAF heterodimerization in dasatinib-induced senescence, a BRAF mutant that prevents dimerization (^R509H5R^F) was transfected into NSCLC cells with endogenous ^wtBRAF either as a single mutation or in cis with ^Y472CBRAF (FIG. 5B) (12). As before, the expression of ^Y472CBRAF led to the activation of ERK and increased sensitivity to dasatinib. The addition of ^R509HBRAF to ^Y472CBRAF inhibited BRAF's ability to activate ERK. NSCLC cells expressing the double mutations were not more sensitive to dasatinib than those expressing ^BRAF (FIG. 5C).

[0070] Drug-induced RAF dimerization alone was not adequate to induce senescence in Call2T or H1666 cells. Nilotinib induced more robust BRAF/CRAF dimerization than did dasatinib in Call2T cells but did not induce senescence (FIG. 15). Together these experiments demonstrate that dasatinib-induced RAF dimerization is essential, but not sufficient for dasatinib sensitivity.

[0071] A preliminary investigation of ERK-independent pathways downstream of RAF demonstrated no mutation-specific changes in BAD or J K (13). Activated Aurora A and PLK1 were not detected by Western blotting (FIG. 16) (14). Src family kinase inhibition with AZD0530 or knockdown with siRNA was not adequate to induce significant senescence or cytotoxicity (FIGs. 15 and 16).

Example 7 - The Combination of a BRAF Inhibitor and Dasatinib Leads to Synergistic

Cytotoxicity in Cancer Cells Resistant to Dasatinib [0072] The sensitivity of cancer cells with inactivating BRAF mutations to dasatinib suggests BRAF's synthetic lethality with a dasatinib target. Cell lines were treated with ^BRAF and marked resistance to dasatinib (15,16) with dasatinib plus the pan-RAF inhibitor sorafenib or dasatinib plus the BRAF inhibitor PLX4032 (vemurafenib) and then measured the treatment's effect on these lines' viability in vitro. In all cases, dasatinib enhanced the effect of the RAF inhibitors at clinically relevant doses (FIGs. 6 and 18) and formal analysis demonstrated synergy. Although specific CRAF and pan-RAF inhibitors may be as effective as dasatinib in treating cancers with inactivating BRAF mutations, no credible direct CRAF inhibitors are currently in clinical development, and pan-RAF inhibitors have poor in vivo activity and are not specific (17, 18). Example 8 - Discussion

[0073] Metastatic NSCLC is a common and fatal disease with a 4-year survival rate of only 2%, but personalized therapies targeting specific genetic aberrations in NSCLC tumors are remarkably successful. A patient was identified with stage IV NSCLC with long term disease control by single-agent dasatinib therapy alone. His tumor harbored a kinase inactivating ^Y472CBRAF mutation that was likely responsible for its unusual sensitivity to dasatinib. Although effective treatments are available for melanoma patients with activating BRAF mutations, no such therapies are available for cancer patients who harbor inactivating mutations.

[0074] No inactivating BRAF mutations were identified in any other patients in the clinical trial, all of whom were nonresponders. As with previously characterized kinase inactivating BRAF mutations, ^Y412CBRAF expression led to CRAF, MEK, and ERK activation. Also, NSCLC cell lines with endogenous inactivating BRAF mutation underwent senescence when exposed to dasatinib; the cell lines' dasatinib sensitivity reversed with the overexpression of active BRAF. Whereas NSCLC cells transfected with an activating BRAF mutation were more resistant to dasatinib than were controls, transfection of NSCLC cells with kinase impaired BRAF led to their increased dasatinib sensitivity. These data and PX's pattern of clinical response are consistent with the conclusion that his tumor underwent senescence when exposed to dasatinib owing to its inactivating BRAF mutation. The mechanism of dasatinib-induced senescence and apoptosis in NSCLC cells expressing kinase impaired BRAF is unknown but may relate to increased RAF dimerization leading to ERK activation consistent with the paradigm of oncogene-induced senescence that occurs following moderate BRAF or KRAS activation (1 1). However, the modest effects on ERK suggest that ERK-independent pathways are involved as well.

[0075] BRAF mutations occur in only about 4% of NSCLC cases but are more common in other tumors, such as melanoma (50%) and papillary thyroid cancer (40%) (19,20). The majority of BRAF mutations cause activation of the kinase (20). In NSCLC, 57% of mutations are remaining 43% are a mixture of kinase inactivating, activating, and uncharacterized mutations (19). CRAF mutations are rare in all cancers (21).

[0076] Interactions between CRAF and BRAF are complex and incompletely understood despite several recent elegant studies in melanoma (4, 5, 22, 23). The pathway is not linear and is further complicated by the multiplicity of signaling molecule components; BRAF and CRAF are similar to each other but do not function identically and are not interchangeable and feedback pathways lead to inactivation of upstream components of the pathway (i.e., a negative feedback amplifier) (24). Additionally, MEK is not the only RAF substrate (25). [0077] The complexity of BRAF-CRAF interactions is epitomized by the RAF inhibitor paradox. Specifically, inhibition of BRAF activity in cells that express ^600EBRAF results in expected inhibition of ERK activity and subsequent apoptosis; melanomas with 6^00EBRAF respond clinically to BRAF inhibition (26). Paradoxically, BRAF inhibition in cells with active RAS leads to ERK activation via activation of CRAF (4). These observations are supported by previous research demonstrating that CRAF and BRAF heterodimers are more active than is either the CRAF or BRAF homodimer even when the BRAF protomer has an inactivating mutation (23).

[0078] These complex interactions of RAS, BRAF, and CRAF explain the existence and function of inactivating BRAF mutations in cancer cells, which superficially seem to be contrary to natural selection. Activation of CRAF not only allows cells expressing kinase impaired BRAF to survive but also may further promote the cancer phenotype by activating non-ERK-dependent pathways or promoting aneuploidy (25, 27). CRAF activation may also be responsible for BRAF-inhibitor-induced squamous cell carcinomas (26). [0079] Although much of the research involving BRAF has focused on melanoma, the BRAF pathway undoubtedly is active and important in NSCLC. RAS is commonly activated in NSCLC via upstream growth factor receptors or activating mutations. Active KRAS signals predominantly through CRAF, not BRAF (28). The role of CRAF in NSCLC has yet to be elucidated, although CRAF is overexpressed in NSCLC cells, and its forced overexpression leads to lung adenomas in a transgenic mouse model (29).

[0080] Oncogene-induced senescence occurs after activation of oncogenes such as RAS and RAF. Classically, it is mediated by activation of the p^^/Rb and/or pi 4^ARF/p53 tumor suppressor pathway. Senescence also can be induced and mediated by other pathways, such as those involving c-Src, STAT3, c-Myc, FOX04, Chk2, and c-Jun-N-terminal-kinase. In the present study, dasatinib induced senescence in HI 666 and Call2T cells but induced apoptosis in H661 cells into which were transfected kinase impaired BRAF mutations. Possible reasons for this discrepancy are that the absolute level of endogenous ^wtBRAF, the level of mutant BRAF, or the w mutant BRAF ratio may influence the outcome of dasatinib- based treatment. The level of mutant BRAF expression was higher in the transfected cells, which also harbored a full complement of endogenous "'BRAF, than in non-trans fected cells. [0081] Occasionally, spontaneous tumor regression occurs in melanoma and renal cell carcinoma cases and is thought to be immune mediated. Also, the activating ^V600EBRAF mutation in melanoma may induce an immune response. Although such a possibility cannot be excluded in the case of PX, spontaneous tumor regression is very rare in NSCLC cases. Instead, the dasatinib sensitivity of NSCLC cell lines with kinase impaired BRAF mutations is consistent with the patient having experienced a direct antitumor effect of dasatinib rather than an immune-mediated mechanism.

[0082] Other potential mechanisms not fully explored in these studies are the roles of other dasatinib targets in mediating dasatinib sensitivity. Although no protein expression has been linked to dasatinib sensitivity, Sos et al. demonstrated that copy-number gains influence sensitivity of NSCLC cells to kinase inhibitors (7). H322 cells, which have c-Src amplification, are sensitive to dasatinib. PX's tumor had copy -number gains in several dasatinib targets. Because live tumor tissue specimens are not available from PX, determining whether his tumor was dependent upon HCK, DDR1, EPHA3, or ARG for survival is impossible (30).

[0083] In conclusion, it was demonstrated that the kinase inactive mutation ^Y472CBRAF functions similarly to other known inactivating BRAF mutations and was likely responsible for PX's response to dasatinib. Moreover, the potential for synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options. Given that dasatinib and BRAF inhibitors are in clinical use, this work has potential for direct clinical applications.

Example 9 - Materials and Methods

Antibodies

[0084] Antibodies used in this study were phosphorylated SFK (pSFK, Y416), pERK 1/2 (T202/Y204), p21^cip, Bcl2, total ERK, pMEK-1/2 (S217/221), phospho-EphA2 (Tyr594), and p-c-Kit (Y719) (Cell Signaling Technology); total CRAF (BD Biosciences); total BRAF, phospho-platelet-derived growth factor (pPDGFRa; Y754), total PDGFRa, c-Kit, Flag M2, and agarose-conjugated CRAF (Santa Cruz Biotechnology); Flag and β-actin (Sigma Chemical Co); and p53 (Dako). HPl-γ (Millipore); Anti mouse Alexa fluor 594 (Molecular Probes). Dasatinib, PLX4032 and sorafenib were purchased from Selleck Chemicals and prepared as 10 mM stock solutions in dimethyl sulfoxide. Cytotoxicity Assay

[0085] Cytotoxicity in NSCLC cells was assessed using a 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described (31). Under each experimental condition, at least four independent wells were treated. Median effects of drugs on viability were calculated using the Chou-Talalay equation (32) with the CalcuSyn software program (Biosoft).

Senescence-Associated β-Galactosidase Staining

[0086] NSCLC cells were processed using a senescence-associated β-galactosdiase staining kit (Cell Signaling Technology) according to the manufacturer's instructions and visualized under an Olympus 1X71 phase microscope (Olympus America). In brief, upon completion of dasatinib-based treatment, cells were washed with phosphate-buffered saline to remove residual media and fixed. A β-galactosidase staining solution containing X-gal was then added to the fixed cells and incubated at 37°C overnight in a dry incubator without CO₂. Fields with at least 100 cells were counted, in triplicate. BRAF and CRAF Kinase Assays

[0087] The kinase activity of immunoprecipitated endogenous BRAF and CRAF protein from NSCLC cells; purified recombinant proteins; or immunoprecipitated Flag- tagged BRAF or Flag-tagged CRAF protein expressed in COS-7 cells was measured using an in vitro kinase assay (IVKA) kit for RAF (Millipore). Purified recombinant BRAF and CRAF (Sigma Chemical Co.) and immunoprecipitated proteins (technique described below using anti-Flag, -HA, -BRAF, or -CRAF antibodies) were incubated with 100-250 μΜ ATP/Mg²⁺ along with 1 μg of inactive recombinant GST-MEK-1 at 30°C for 30 min and subsequently boiled with lx sample buffer to stop the reaction. MEK-1 activation was quantified by measuring the phospho-specific MEK-1 band (ImageJ software program; National Institutes of Health) after Western blotting (described below).

Cell-Cycle, Proliferation and Apoptosis Assays

[0088] For cell-cycle analysis, cells were harvested, fixed, and stained with propidium iodide, and their DNA content was analyzed using a cytofluorimeter and fluorescence-activated cell sorter (FACS) (FACScan; Becton Dickinson) and using the ModFit software program (Verity Software House) (31). To measure apoptosis, fixed cells were subjected to terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TU EL) staining according to the manufacturer's procedure (APO-BRDU kit; Phoenix Flow Systems) and quantitated using the FACS (31). BrdU incorporation was measured according to the manufacturer's instruction (BrdU Flow Kits, BD Biosciences). Briefly, subconfluent cell cultures were treated with DMSO or 150 nM dasatinib for 72 hours, then labeled with 10 μΜ BrdU for 4 hr. Cells were trypsinized, fixed, and stained with FITC- conjugated anti-BrdU antibody and 7-Aminoactinomycin D (7-AAD). Samples were analyzed by two-dimensional flow cytometry to detect both fluorescein and 7-AAD.

Transfection of NSCLC Cells with a Small Interfering RNA [0089] Cells were harvested, washed, and suspended (10⁶ cells/ 100 μΐ,) in

Nucleofector Solution V (Amaxa), and small interfering RNA (siRNA; 200 pmol/100 μί) was added to the cells. Cells were then electroporated using the Nucleofector program U24 (Amaxa) and diluted with a prewarmed 500-μΕ RPMI medium supplemented with 10% serum and plated onto 60-mm plates and the medium was changed 16 hours later. The c-Src and CRAF siRNAs were predesigned as sets of four independent sequences (siGENOME SMARTpool; Dharmacon). Controls were cells transfected with a nontargeting (scrambled) siRNA and mock-transfected cells (i.e., no siRNA).

Site-Directed Mutagenesis

[0090] Flag-tagged ^BRAF and ^600EBRAF plasmids were provided by Dr. Walter Kolch (Systems Biology Ireland and The Conway Institute, University College Dublin). The ^BRAF construct was used as a template to create the ^Y472CBRAF , ^G466 BRAF, ^R509HBRAF and ™ ™∞_{BRAF mutations} using the QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The sense mutagenic primers used were 5'-GATCATTTGGAACAGTCTGCAAGGGAAAGTGGCATGGT -3' (SEQ ID NO: 6) for ^Y412CBRAF and 5'-GTGGGACAAAGAATTGGATCTGTATCATTTGGAACA GTC -3' (SEQ ID NO: 7) for ^G466W BRAF, 5'-

GTAGGAGTACTCAGGAAAACACACCATGTGAATATCCTACTCT-3 ' (SEQ ID NO:8) for ^R509HBRAF and ^Υ472™⁵⁰⁹¾/^ _{To con}firm successful introduction of the mutations, six different plasmids for each mutant were Sanger sequenced. Immunoprecipitation and Western Blot Analysis

[0091] For both Western blot and immunoprecipitation (IP) analysis, cells were lysed on ice, and the lysates were centrifuged at 20,000g for 5 min at 4°C as described previously (31). For IP, equal amounts of the protein cell lysate supernatant (500 mg) were precleared with protein A and G sepharose beads (Invitrogen). The precleared lysate was incubated with an IP antibody (anti-Flag or anti-CRAF) overnight. The immunoprecipitates were washed four times with an immunocomplex wash buffer (50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 1% Triton X-100, 1 mmol/L egtazic acid, 1 mmol/L ethylenediaminetetraacetic acid, 1% glycerol, 20 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium vanadate) and boiled with l x sample buffer for 5 min. For both the IP and Western blot analysis, equal protein aliquots were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, immunoblotted with a primary antibody, and detected using a horseradish peroxidase- conjugated secondary antibody (Bio-Rad Laboratories) and ECL reagent (Amersham Biosciences).

Clinical Tissue Specimens

[0092] Patients' tissue specimens were obtained in an MD Anderson Institutional Review Board-approved clinical trial in which all participants gave permission for testing of residual tumor tissue. For PX, residual tissue from a metastatic axillary lymph node resected 2 months before treatment was used. The metastasis was formalin-fixed and paraffin- embedded (FFPE) according to a standard protocol. One 4-μιη tissue section was cut and stained with hematoxylin and eosin. Histopathological tumor examination and visual estimation of the tumor area and tumor-cell percentage were conducted by a pathologist. The tissue section had a 2.5 x 1.8 cm lymph node extensively infiltrated with malignant epithelial cells representing -60 % of the whole tissue section. Five 10-μιη unstained tissue sections were cut and placed in sterile tubes for genomic DNA (gDNA) extraction and isolation.

Cell Culture

[0093] Call2T cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. All other human NSCLC cell lines were obtained from Drs. John Heymach (The University of Texas MD Anderson Cancer Center) and John Minna (The University of Texas Southwestern Medical Center) and maintained using standard cell culture techniques (30). COS7 monkey kidney cells were obtained from Dr. Ho-Young Lee (MD Anderson). All cell lines were validated by cross-comparing their allelic short tandem repeat patterns generated using the PowerPlex 1.2 system (Promegl) with the American Type Culture Collection repository database. BRAF mutations were confirmed using Sanger sequencing of exons 1 1 and 15.

Immunofluorescence microscopy

[0094] Cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.5% NP40 for 10 min at room temperature. Cover slips were blocked in 10% normal goat serum in PBS for 30 min, and incubated with the anti-ΗΡΙγ antibody (1 :200 dilution). Cells were then washed and incubated with the anti-Alexa Fluor 594 antibody. After washing, cells were incubated with DAPI for 30 min, washed again and then the cover slips were mounted on the slides. Confocal microscopy was performed using an Olympus IX- 81 Spinning Disc Confocal microscope using a 60x water immersion 1.2 NA objective and 31 Slidebook 5.0 software. Copy-Number-Variation Analysis

[0095] Copy-number-variation analysis of PX's tumor was conducted using a human genome CGH microarray kit (244A; Agilent Technologies). This platform uses 240,000 unique 60-mer oligonucleotide probes across the genome, with tighter coverage in the regions of RefSeq genes. Array hybridization was performed using 2.5 μg of gDNA from PX's tissue specimen and a control human gDNA (Promega) according to the array manufacturer's protocol. Briefly, hybridization lasted for 40 h at 65°C, and hybridized arrays were scanned using an Agilent Technologies dual-laser-based scanner. Data transformation was performed using the Feature Extraction CGH-v4_91 software program (Agilent Technologies). Statistical aberration detection was conducted using the Nexus Copy Number software program (version 5.0; BioDiscovery, El Segundo, CA).

MassARRAY Mutation Analysis

[0096] gDNA from the specimens was analyzed using polymerase chain reaction (PCR) amplification followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy single-nucleotide polymorphism analysis (MassARRAY; Sequenom, Inc., San Diego, CA) for 40 genes with 240 mutations (Table 1). PCR products were classified as wt or mutant according to their molecular weights. Detected mutations were then Sanger sequenced.

Primers for Mutational Analysis of BRAF and c-Src

[0097] The primers used for c-Src were CTT CTC CTT TCC TCC CTC CTT (forward; SEQ ID NO:9) and CAG GAG AGG CAC TCT GCAC (reverse; SEQ ID NO: 10) for exon 7, AGC CAT ATC CAG GGA GAA GC (forward; SEQ ID NO: l 1) and ACA CCC AGC TCA AAC CAC TC (reverse; SEQ ID NO: 12) for exon 8, CCT TCC CTC CAA TGT CAG G (forward; SEQ ID NO: 13) and AGT CTG CAG CTG AGG CTT TG (reverse; SEQ ID NO: 14) for exon 9, and AGA AGA CCC GCC TAA CTG CT (forward; SEQ ID NO: 15) and ATC CAG CAG AGG CAG CTA AAG (reverse; SEQ ID NO: 16) for exon 10. The primers used for BRAF were TCC CTC TCA GGC ATA AGG TAA (forward; SEQ ID NO: 17) and CGA ACA GTG AAT ATT TCC TTT GAT (reverse; SEQ ID NO: 18) for exon 11 and TCA TAA TGC TTG CTC TGA TAG GA (forward; SEQ ID NO: 19) and GGC CAA AAA TTT AAT CAG TGG A (reverse; SEQ ID NO:20) for exon 15. Mutational Analysis of BRAF and c-Src

[0098] Intron-based PCR was used to sequence B-Raf exons 1 1 and 15 and c-Src exons 7-10 as described previously. FFPE tumor tissue was microdissected, and about 200 cells were used in each amplification. DNA extracted from either microdissected tissue or cell pellets was subjected to PCR, and PCR products were directly sequenced using a PRISM dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). The primers are listed in the supplemental methods. Sequence variants were confirmed using independent PCR amplifications and sequenced in both directions

DDR2 Sequencing

[0099] DDR2 mutations within coding exons were screened from genomic DNA obtained from patient's tumor sample using Sanger sequencing method as described (6). The coding exons were designated based on mRNA transcript variant 2 from DDR2 gene (NM_ 006182). Mutations( based on SNP locations) were verified manually with comparison made to the matched normal sequence. Fluorescence In Situ Hybridization

[00100] A fluorescence in situ hybridization (FISH) assay following a standard protocol was performed to determine the presence of an ALK gene rearrangement using the LSI ALK dual-color break-apart probe (Abbott Molecular, Abbott Park, IL). PX's tissue slides were incubated for 4 hours at 56°C, deparaffinized in Citri-Solv (Fisher), and washed in 100% ethanol for 5 min. The slides were pretreated using a Dako histology FISH accessory kit (K5599) in a pressure cooker as follows: at 121°C for 1 min, cool-down to 90°C, then at room temperature for 15 min. Excess liquid was removed and the slides were incubated in Dako pepsin at 37°C for 2 hours. Next, the slides were washed in Dako wash buffer and dehydrated in ethanol. The probe sets were applied to the tissue which was covered with a coverslip and sealed with rubber cement. The specimen was denatured for 8 min at 80°C and incubated at 37°C for 48 h. Posthybridization washes were performed using a wash buffer (1.5 M urea in O. lx standard sodium citrate). The slides were then washed in 2x standard sodium citrate for 2 min and dehydrated in ethanol. Chromatin was counterstained with DAPI (0.3 μg/mL in Vectashield; Vector Laboratories, Burlingame, CA). Analysis was performed using a fluorescence microscope with single interference filter sets for green (SpectrumGreen), orange (SpectrumOrange), and blue (DAPI) bandpass filters. Monochromatic images were captured and merged using a CytoVision workstation (Applied Imaging, San Jose, CA). Cells with single or split signals were defined as those with gene rearrangement. Those with both signals close or overlapping were negative for rearrangement.

Genomic DNA Preparation

[00101] Total nucleic acids were extracted from five tissue sections using an SPRITE total nucleic acid extractor (Beckman Coulter). Each section was individually extracted and gDNA isolated. Briefly, sections were incubated in 200 of lysis buffer for 1 hour at 85 °C followed by 30 of proteinase K for 1 hour at 55°C, producing a total nucleic acid fraction. Next, gDNA was isolated from the sections and cleaned for array comparative genomic hybridization (aCGH) and matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy single-nucleotide polymorphism analysis using a standard column cleanup with RNase treatment to remove RNA from the total nucleic acid fraction (DNeasy blood and tissue kit; QIAGEN) and adapted from an FFPE DNA preparation for oligonucleotide array- based CGH for a gDNA analysis protocol (Agilent Technologies). gDNA quantity and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific). The mean gDNA quantity recovered per tissue section was 10,340 ng. The mean sample purity (1.81) as assessed using a 260:280 wavelength ratio was within accepted DNA-purity levels (28). Individual gDNA preparations from each section were then pooled for use. Immunohistochemistry

[00102] An automated stainer (Dako) was used for immunohistochemical staining of tumor sections. Five-micrometer sections of FFPE tumor were deparaffinized and hydrated, and antigen retrieval was done (pH 6.0, Dako). Cell pellets from NSCLC cell lines with high and low protein expression levels according to Western blotting relative to the levels in a panel of eight NSCLC cell lines were used as positive and negative controls for the staining. Peroxide blocking was performed using 3% methanol and hydrogen peroxide for 15 min. Upon blocking with 10% fetal bovine serum, tissue slides were incubated with the primary antibodies phospho-PDGFRa (Y754, 1 :500), total PDGFRa (1 :500), c-Kit (1 :200), EphA2 (1 :500), phospho-EphA2 (Tyr594, 1 :500), phospho-c-Kit (Y719, 1 : 150), and p53 (1 :400) at room temperature for 1 hour. After washing in Tris-buffered saline and Tween 20, slides were incubated with Dako EnVision+ Dual Link reagent for 30 min at room temperature followed by a Dako chromogen substrate for 5 min and were then counterstained with hematoxylin for 5 min. Slides were examined for the staining intensity by a blinded observer using a light microscope with a *20 magnification objective. Cytoplasmic staining was scored based on both staining intensity (0-3 scale: 0, below the level of detection; 1 , weak; 2, moderate; 3, strong) and the percentage of cells stained at each intensity level (0- 100%). The final score was calculated by multiplying the intensity score by the percentage, producing a scoring range of 0-300.

* * * [00103] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

1. An in vitro method for identifying a candidate for a Src inhibitor therapy comprising:

(a) testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein; and

(b) identifying the subject as a good candidate for a Src inhibitor therapy if the sample from the subject indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity; or identifying the subject as a poor candidate for a Src inhibitor therapy if the sample from the subject does not indicates that cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity.

2. A method for identifying a good candidate for a Src inhibitor therapy comprising testing a biological sample from a subject having a cancer to determine whether cells of the cancer comprise a BRAF gene encoding a protein with reduced kinase activity relative to a wild type BRAF protein, thereby identifying the subject as a good candidate for a Src inhibitor therapy.

3. The method of claim 1, wherein testing the sample comprises determining whether cells of the cancer comprises a BRAF gene encoding a protein with reduced kinase activity.

4. The method of claim 3, wherein the BRAF gene encodes a protein comprising an amino acid substitution or deletion at position Y472.

5. The method of claim 4, wherein the BRAF gene encodes a protein comprising a Y472C amino acid substitution.

6. The method of claim 1, wherein testing the sample from the subject comprises determining all or part of a BRAF gene sequence in the sample.

7. The method of claim 1, wherein testing the sample from the subject comprises measuring BRAF kinase activity in the sample.

8. The method of claim 1, wherein the sample comprises cancer cells.

9. The method of claim 1, wherein a good candidate for a Src inhibitor therapy comprises a subject that is predicted to have reduced in tumor size or burden, reduced tumor growth, reduced tumor-associated pain, reduced cancer associated pathology, reduced cancer associated symptoms, cancer non-progression, cancer remission, reduced cancer metastasis, or increased survival time in response to a Src inhibitor therapy.

10. The method of claim 1, wherein the cancer is a lung cancer.

11. The method of claim 10, wherein the lung cancer is a non-small cell lung cancer.

12. The method of claim 10, wherein the cancer is a metastatic lung cancer.

13. The method of claim 1 or 2, wherein the Src inhibitor therapy is a Src kinase inhibitor.

14. The method of claim 13, wherein the Src kinase inhibitor is bosutinib, saracatinib, danusertib, NVP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib.

15. The method of claim 14, wherein the Src kinase inhibitor is dasatinib.

16. The method of claim 1, further comprising providing a report indicating whether the subject is a good candidate or a poor candidate for a Src inhibitor therapy.

17. The method of claim 16, wherein the report is a written or electronic report.

18. The method of claim 16, wherein providing a report comprises transmitting the report to a doctor or a hospital.

19. A method for treating a subject having a cancer, wherein it was determined that cancer cells from the subject comprise reduced BRAF kinase activity relative to a control level, the method comprising administering a Src inhibitor therapy to the subject.

20. The method of claim 19, wherein the cancer is a lung cancer.

21. The method of claim 20, wherein the lung cancer is a non-small cell lung cancer.

22. The method of claim 20, wherein the cancer is a metastatic lung cancer.

23. The method of claim 19, wherein the Src inhibitor therapy is a Src kinase inhibitor.

24. The method of claim 23, wherein the Src kinase inhibitor is bosutinib, saracatinib, danusertib, NVP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib.

25. The method of claim 24, wherein the Src kinase inhibitor is dasatinib.

26. The method of claim 19, wherein it was determined that cancer cells from the subject comprise a BRAF gene encoding a protein with reduced kinase activity.

27. The method of claim 26, wherein the BRAF gene encodes a protein comprising an amino acid substitution or deletion at position Y472.

28. The method of claim 27, wherein the BRAF gene encodes a protein comprising a Y472C amino acid substitution.

29. The method of claim 19, wherein the Src inhibitor therapy is administer two or more times.

30. The method of claim 19, further comprising administering at least a second anticancer therapy to the subject.

31. The method of claim 30, wherein the second anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or cytokine therapy.

32. A composition for the treatment of a subject having cancer comprising cancer cells with reduced BRAF kinase activity relative to a control level, the composition comprising a therapeutically effective amount of a Src inhibitor.

33. A tangible computer-readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform operations comprising:

a) receiving information corresponding to a BRAF kinase activity in a sample from a subject having a cancer; and

b) determining a relative level of BRAF kinase activity compared to a reference level, wherein reduced BRAF kinase activity relative to the reference level indicates the subject is a good candidate for a Src inhibitor therapy.

34. The method of claim 33, wherein the information corresponding to a BRAF kinase activity comprises all or part of a BRAF gene sequence.

35. The method of claim 34, wherein determining a relative level of BRAF kinase activity comprises determining a change in all or part of a BRAF gene sequence compared to a reference sequence, wherein a change in the BRAF gene sequence corresponding to reduced BRAF activity indicates that the subject is a good candidate for a Src inhibitor therapy.

36. The method of claim 34, wherein the reference level is all or part of a wild type BRAF gene sequence.

37. The tangible computer-readable medium of claim 33, wherein the receiving information comprises receiving from a tangible data storage device information corresponding to a level of BRAF kinase activity in a sample from a subject having a cancer.

38. The tangible computer-readable medium of claim 33, further comprising computer- readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the relative level of BRAF kinase activity to a tangible data storage device.

39. The tangible computer-readable medium of claim 33, wherein the computer-readable code that, when executed by a computer, causes the computer to perform operations further comprising: c) calculating a diagnostic score for the sample, wherein the diagnostic score is indicative of the probability that the subject will respond to a Src inhibitor therapy.

40. A method for treating a subject having a cancer comprising administering to the subject an effective amount of a Src inhibitor in conjunction with a RAF inhibitor.

41. The method of claim 40, wherein the Src inhibitor is a Src kinase inhibitor.

42. The method of claim 41, wherein the Src kinase inhibitor is bosutinib, saracatinib, danusertib, NVP-BHG712, quercetin, PCI-32765, KX2-391, AP23451 or dasatinib.

43. The method of claim 42, wherein the Src kinase inhibitor is dasatinib.

44. The method of claim 40, wherein the RAF inhibitor is a BRAF specific inhibitor

45. The method of claim 40, wherein the RAF inhibitor is vemurafenib, GSK2118436 or sorafenib.