WO2022006292A1

WO2022006292A1 - Nek2 proteolysis targeting chimeras for use in malignant disease

Info

Publication number: WO2022006292A1
Application number: PCT/US2021/039923
Authority: WO
Inventors: Brendan FRETT; Samantha KENDRICK
Original assignee: Bioventures, Llc
Priority date: 2020-06-30
Filing date: 2021-06-30
Publication date: 2022-01-06

Abstract

This invention describes a small molecule proteolysis targeting chimera (PROTAC) strategy to degrade the NEK2 (Never in Mitosis Related Kinase 2) protein and methods of use thereof. The NEK2 PROTAC consists of three, key regions: a NEK2 ligand to bind the NEK2 protein, a linker region to link the NEK2 ligand to a ligase ligand, and the ligase ligand.

Description

NEK2 PROTEOLYSIS TARGETING CHIMERAS FOR USE IN MALIGNANT DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of United States Provisional Patent Application Ser. No. 63/046,209, filed June 30, 2020, the contents of which is incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includes an electronically submitted Sequence Listing in .txt format. The .txt file contains a sequence listing entitled "169852_00082_ST25.txt" created on June 30, 2021 and is 1.67 KB bytes in size. The Sequence Listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.

BACKGROUND

Current chemotherapy places adolescent and young adults (AY A) with diffuse large B- cell lymphoma (DLBCL) at high risk for acute and chronic toxicities and secondary cancers. There is an elevated risk for AYA lymphoma patients to develop chronic disease and secondary malignancies in response to currently used chemotherapy. Unlike adults diagnosed with DLBCL, AYA patients typically achieve a 5-year survival rate of 90% ^{L6 7}. Despite the curative outcomes, there is a high risk for treatment induced toxicity and secondary cancers post-therapy. AYA patients receive the same chemotherapy cocktail of anthracycline, steroid hormone, DNA alkylating and microtubule disrupting agents, but often without rituximab, an anti-CD20 monoclonal antibody incorporated into the adult regimen over 15 years ago. Ongoing clinical trials with young non-Hodgkin’s lymphoma patients, including those with DLBCL, are investigating the efficacy of rituximab. These initial studies show around one-third of children and adolescent patients (<18 years) suffer from grade 4 - 5 acute adverse events, where grade 5 is death, but overall and progression-free survival is improved⁸. These recent findings are consistent with a previous study using age/sex-matched healthy individuals as controls that found AYA non-Hodgkin’s lymphoma survivors more frequently experienced grade 1 - 5 chronic health conditions². The devastating, latent toxicity observed in AYA cancer patients emphasizes the need for targeted therapies to lower the risk of the morbidity associated with harsh, non specific chemotherapy. As a result, there exists a need for new therapeutic approaches that target these distinct characteristics to improve quality of survivorship

SUMMARY OF THE INVENTION

Disclosed herein is a proteolysis targeting chimera (PROTAC) strategy to degrade the NEK2 (Never in Mitosis Related Kinase 2) protein. The NEK2 PROTAC consists of three, key regions: a NEK2 binding ligand to bind the NEK2 protein, a linker region to link the NEK2 ligand to a E3 ligase ligand, and the E3 ubiquitin ligase ligand. The present technology utilizes a PROTAC strategy to completely degrade the NEK2 oncoprotein. Prior methods rely on simply blocking catalytic NEK2 activity while the oncoprotein remains intact in the tumor. Completely degrading the NEK2 oncoprotein will impair catalytic as well as scaffolding proprieties to eliminate all protumor aspects of the oncogene. Completely degrading the NEK2 oncoprotein can serve as a therapeutic method to better treat malignancies that depend on the oncoprotein.

One aspect of the invention provides for a proteolysis targeting chimera of formula LL-L- NBL where NBL is a NEK2 binding ligand, LL is a ligase ligand, and L is a linker-region to link the NEK2 ligand to the ligase ligand. In some embodiments, the NEK2 binding ligand is a Ne-1 moiety. In some embodiment, the ligase ligand is an ubiquitin ligase ligand, such as thalidomide, lenalidomide, avadomide, or VH 032. In some embodiments, the linker region comprising 1-5 ethylene glycol units. In a particular embodiment, the proteolysis targeting chimera is a compound of formula

Another aspect of the invention provides for pharmaceutical compositions comprising any of the proteolysis targeting chimeras described herein. In some embodiments, the composition further comprises an anti-cancer agent.

Another aspect of the invention provides for a method for treating of subject in need of a proteolysis targeting chimera. The method may comprise an effective amount of any of the proteolysis targeting chimeras described herein.

In some embodiments, the subject is in need of the proteolysis targeting chimera for treating a cancer. The cancer may be a DLBCL, including AYA DLBCL or ABC DLBCL.

In some embodiments, the subject is in need of the proteolysis targeting chimera comprising for sensitizing the subject to an anti-cancer agent. The method may further comprise administering an effective amount of the anticancer agent. In some embodiments, the subject is in need of a treatment for a cancer, such as DLBCL, including AYA DLBCL or ABC DLBCL.

Another aspect of the invention provides for a method for proteolysis of a NEK2 protein comprising contacting the NEK2 protein. The method may comprise contacting the NEK2 protein with any of the proteolysis targeting chimeras described herein. In some embodiments, the EK2 protein is contacted with the proteolysis targeting chimera in a cell. The cell may be a cancer cell. Suitably, the cancer cell is a DLBCL cancer cell, including AYA DLBCL cancer cells or ABC DLBCL cancer cell.

Another aspect of the invention provides for a method for inhibiting the growth or proliferation of a cell or for killing the cell. The method may comprise contacting the cell with an effective amount of any of the proteolysis targeting chimeras described herein. The cell may be a cancer cell. Suitably, the cancer cell is a DLBCL cancer cell, including AYA DLBCL cancer cells or ABC DLBCL cancer cell.

Another aspect of the invention provides for a method for sensitizing a cell to an anti cancer agent. The method may comprise contacting the cell with an effective amount of any of the proteolysis targeting chimeras described herein. The method may further comprise contacting the cell with an effective amount of the effective amount of the anti-cancer agent. Suitably, the cancer cell is a DLBCL cancer cell, including AYA DLBCL cancer cells or ABC DLBCL cancer cell.

These and other aspects of the invention will be further described herein. BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

Figure 1 illustrates that NEK2 is an essential component of the intercentriolar linkage, aids in centrosome localization of MAPK1, dysregulates phosphatase PP1, and has a signaling loop with CDK4 (17, 19, 46, 47). Catalytic, scaffolding, and protein-protein interactions all contribute to the oncogenicity of NEK2.

Figure 2 illustrates a selective, NEK2 scaffold (Ne-1) to generate NEK2 PROTACs. The linker and E3 ligand may be attached from Position 1 or, alternatively, Positions 2 and 3.

Figure 3 shows that the selective, NEK2 inhibitor, Ne-1, exhibits high selectivity for the ABC subtype of DLBCL.

Figure 4 shows (A) computational modeling of Ne-1 in NEK2 with the pyrazole oriented towards the solvent (arrow) and (B) Ne-l/PROTAC modeled in the NEK2 protein. The PROTAC can freely bind to the E3 ligase when bound to NEK2.

Figure 5 illustrates an exemplary NEK2 PROTAC based on Ne-1 and demonstrates variability in the type of linker and E3 ligase ligand installed.

Figure 6 illustrates (A) a synthetic scheme for preparing INT2-Ne-1 where reaction conditions may include a) Pd (dba)₃, P(Cy)₃, Na CCh, DMF/H O (4:1), Boronic Acid, and 120 °C and b) TFA, DCM, 0 °C to RT. Figure 6 also illustrates (B) different PROTAC linkers with differing number of units may be installed to prepare NEK2 PROTACs.

Figure 7 illustrates exemplary E3 ligase ligands with functional group indicated for coupling to a linker region.

Figure 8 shows AYA DLBCL exhibit a distinct gene expression pattern of NEK2 regulated pathways. (A) Heatmap of differentially expressed genes using Affymetrix U133 Plus 2 chip (n = 32 for each cohort). (B) Box plot showing median and range of NEK2 protein expression using immunohistochemistry (n = 9 for each cohort). Representative light micrographs of NEK2 staining in brown from DLBCL derived from a young adult and an adult patient. Samples from (A) and (B) are pair-matched for sex, stage, IPI, and GCB/ABC subtype prior to analyses ’-value determined by Student’s t-test with Welch’s correction for significant differences in standard deviation. Scale bar = 20 pm.

Figure 9 shows AYA DLBCL express a positive cell cycle and centrosome separation profile. (A) Heatmap of differentially expressed proteins identified by TMT-MS of AYA (SUDHL5) and adult (VAL) DLBCL cells. Triplicate samples (lanes 1-3 and 4-6). (B) Fold- change of EK2 regulated cell cycle and centrosome related proteins. (C) EGSEA showed age related differences in pathways expressed in these two female derived cell lines demonstrating the sensitivity of the proteomic analyses and the possibility that the cell lines retained some of their original patient characteristics despite maintenance in culture.

Figure 10 shows NEK 2 inhibition extensively alters the proteome landscape. (A) Heatmap of the differentially expressed proteins identified by TMT-MS of SUDHL5 AYA DLBCL cells in the absence and presence of the NEK2 inhibitor. Triplicate samples (lanes 1-3 and 4-6). (B) Key proteins identified. (C) Corresponding fold-change in phosphorylation of proteins when NEK2 activity is inhibited. (D) Consensus motifs for altered phosphorylation sites.

Figure 11 shows sensitization of AYA DLBCL cells to chemotherapy. (A) AYA and (B) adult DLBCL, and (C) benign B-cells were co-treated with increasing concentrations of the NEK2 kinase inhibitor and static, non-cytotoxic concentrations of DOX (doxorubicin) or VCR (vincristine). The highest concentration of the NEK2 inhibitor (7.8 nM) served as a control to demonstrate there is no decrease in cell viability from this treatment alone. (D) Growth inhibitory concentrations (GCso) for each drug /i- values were adjusted using Sidak’s multiple comparisons test: *p < 0.05; **p < 0.001; ***p < 0.0001.

Figure 12 shows NEK2 PROTAC has greater impact on downstream NEK2 substrates. (A) Western blot confirming lower NEK2 protein levels after treatment with the PROTAC. (B) Total- and (C) phosphorylated proteins identified by TMT-MS that were differentially expressed compared to DMSO vehicle-treated control after treatment with either the NEK2 inhibitor or PROTAC. Data in (A-C) were collected from AYA SUDHL5 cells. DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are proteolysis targeting chimeras (PROTACs) and methods of making and using the same. PROTAC technology is a powerful technique to selectively position proteins for complete inactivation. PROTACs allow for the preparation of bifunctional molecules that can inhibit enzymatic function and proteolytically degrade target proteins, such as oncoproteins like NEK2. The presently disclosed technology can reduce the potential for drug resistance, improve target selectivity, or lower effective drug concentrations.

A major clinical limitation to precision medicine is resistance due to target mutation, signal-rewiring to bypass inhibition, and/or upregulation of the target protein. Proteolysis targeting chimeras, or PROTACs, are bifunctional small molecules that can be exploited in precision medicine to selectively degrade an oncoprotein and substantially lower potential for drug resistance and relapse, which is a clinical liability for current inhibitors.

Another clinical limitation to cell-cycle kinase inhibitors is a lack of a therapeutic window relative to benchmark treatments such as paclitaxel or vinblastine. Although cell-cycle kinases are overexpressed or upregulated in many human cancers, their targeting is contraindicated due to dose-limiting toxicities and strict dosing schedules to maintain active concentrations of the drug throughout the cell cycle.

To better identify approaches to combat human malignancies, the presently disclosed PROTACS eliminate protein levels of cell-cycle kinases exploited by cancers. While protein kinases are druggable, kinase-based PROTACs (KBPs) have longer duration of action and require lower concentrations than compounds that only inhibit catalytic activity.

The PROTACs of the present invention comprise a compound of formula LL-L-NBL where NBL is a NEK2 binding ligand, LL is a ligase ligand, and L is a linker-region to link the NEK2 binding ligand to the ligase ligand. The NEK2 binding ligand is a protein target moiety. A “protein target moiety” is a small molecule that binds to a target protein or other protein or polypeptide of interest and places/presents that protein or polypeptide in proximity to a ligase, such as an ubiquitin ligase, such that degradation of the protein or polypeptide may occur. Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates and polymorphs of these compositions, as well as other small molecules that may target a protein of interest. "Target protein” is used to describe a protein or polypeptide, such as a NEK2 protein, which is a target for binding to a compound according to the present invention and degradation by ubiquitin ligase hereunder. A "NEK2 protein" refers to proteins that have partial sequence homology (e.g., at least 5%, 10%, 25%, 50%, 75%, 85%, 95%, 99%, 99.999%) or complete sequence homology with NEK2 and which is capable of binding to the NEK2 binding ligand. In some embodiments, the NEK2 binding ligand is a moiety or functional group having binding affinity for a NEK2 protein and may be capable of binding into the active site of a NEK2 protein. Suitably, the NEK2 binding ligand may inhibit catalytic activity of a NEK2 protein.

The Nek family of serine/threonine kinases are essential for mitosis and cell cycle regulation. The best characterized member of the Nek family is NIMA (Never In Mitosis/Aspergillus), which is crucial for entry into mitosis and highlights the target as a potential mitotic inhibitor. The closest related mammalian isoform to NIMA is NEK2 (NIMA Related Kinase 2). During the G2/M stage of the cell cycle, NEK2 initiates centrosome separation; thus, deficiencies in NEK2 activity leads to G2/M arrest and over activity of NEK2 bypasses the G2/M checkpoint. As shown in Figure 1, NEK2 phosphorylates a variety of substrates, including b-catenin and serine/arginine-rich splicing factor 1 (SRSF1), which maintains pro-tumor signaling in the cell. NEK2 is also an essential component of the intercentriolar linkage, aids in the centrosome localization of MAPKl, forms signaling loops with CDK4, and jeopardizes the cellular regulation of AKT. Overexpression of NEK2 promotes premature separation of chromosomes at the intercentriolar linkage, yielding daughter cells with high amounts of aneuploidy. Therefore, aberrant NEK2 is detrimental for the accurate transfer of genetic information. Elevated levels of NEK2 enhance AKT activity via phosphatase dysregulation. Consequently, NEK2 represents an essential component of the intercentriolar linkage and augments the activity of key oncogenic pathways, such as AKT. Therefore, its kinase activity, and its scaffolding role as a critical component of the intercentriolar linkage, suggest degradation of NEK2 is optimal to remove catalytic and scaffolding properties.

Due to the multifaceted nature of NEK2, its catalytic ability, scaffolding, and protein- protein interaction properties all contribute to pro-tumor phenotypes. For example, ABC DLBCL has increased endogenous expression of NEK2 compared to GCB, and ABC DLBCL is more sensitive to NEK2 perturbation than the GCB subtype. As a result, it is possible to generate NEK2 PROTACs with heightened activity and selectively degrade NEK2 in cancer or tumor cells.

In some embodiments, the NEK2 binding ligand comprises a Ne-1 moiety. Ne-1 (Figure 2) is a potent and selective NEK2 inhibitor. Ne-1 exhibits an ICso of 30 nM against NEK2 with selectivity against a 97 kinase panel representing all kinase clusters. The selective NEK2 scaffold is a Type-I kinase inhibitor and exhibits reversible binding kinetics. Reversible, binding kinetics are important for the reuse of PROTAC -based small molecules after protein degradation to permit catalytic, drug concentrations for efficient protein degradation. Other NEK2 binding ligands may also be used, including, without limitation, those disclosed in WO 2018/081719 to Li el al.

Ne-1 exhibits exquisite selectivity for the aggressive activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) compared to the more chemo-sensitive germinal center B-cell (GCB) subtype. As shown in Figure 3, Ne-1 is 100 times more active on ABC DLBCL compared to the GCB subtype. With this selectivity, NEK2 KBPs can be developed to remove the NEK2 oncoprotein with low effective drug concentration, increase duration of action, and improve efficacy profiles. Moreover, eliminating NEK2 protein in ABC DLBCL and other cancer cells can provide tissue-specific loss of function of an oncoprotein.

Like other kinase active sites, the NEK2 active site is solvent exposed, which permits the facile attachment of ligase ligands. Based on computational modeling experiments with Ne-1 (Figure 4), the aliphatic, dimethylamine orientation is towards the solvent pocket of NEK2. In the active site, the dimethylamine does not form any key interactions with the NEK2 kinase and sits in the solvent. Therefore, this location is well suited for modification to incorporate a linker- region with a ligase ligand. A linker-region with a ligase may also be installed at other positions in the Ne-1 ligand. In some embodiments, the linker-region may be installed on the imidazopyridine ring system. For example, linkers may be installed at carbon 6 or 5 (Figure 2). In other embodiments, the linker-region with a ligase may be install at the "selectivity region" at Position 3 (Figure 2).

Figure 5 illustrates an exemplary NEK2 PROTAC comprising a Ne-1 moiety. The NEK2 PROTAC comprises linker comprising ethylene glycol units and an unbiguitin ligase ligand comprising a thalidomide moiety. As further described below, other linkers or ligase ligands may be employed. The NEK2 binding ligand and the ligase ligand may be chemically linked or coupled via a chemical Linker (L). The linker should allow for appropriate formation of a target protein- ligase ternary complex. The linker group may comprises one or more structural units A, such as ethylene glycol (-CH2CH2O-) or alkylene (-CH2) units.

In some embodiments, the linker may be a polyethylene glycol chain which may terminate (at either or both termini) in at least one of-P(0)(OH)0-, -S-, -N(R')-, -C(O)-, -C(0)0-,

N(R)C(0)-, -N(R')C(0)N(R)-, -N(R)C(0)0-, -OC(0)N(R)-, -C(NR , -N(R)C(NR , - C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O) 2- , -OS(O)-, -S(0)0-, -S(O)-, -OS(0)₂- -S(O)₂0-, - N(R)S(0)₂- , -S(0)₂N(R)-, -N(R')S(0)-, -S(0)N(R)-, -N(R)S(0)₂N(R , -N(R)S(0)N(R')-, C3- 12 carbocyclene, 3- to 12-membered heterocyclene, 5-to 12-membered heteroarylene or any combination thereof, wherein R is H or C1-C6 alkyl, wherein the one or both terminating groups may be the same or different. In some embodiments, a polyethylene glycol chain may comprise

1-10 ethylene glycol units, 1-9 ethylene glycol units, 1-8 ethylene glycol units, 1-7 ethylene glycol units, 1-6 ethylene glycol units, 1-5 ethylene glycol units, 1-4 ethylene glycol units, 1-3 ethylene glycol units, 1-2 ethylene glycol units, or 1 ethylene glycol unit.

In some embodiments, the linker may be an alkylene chain or a bivalent alkylene chain, either of which may be interrupted by, and/or terminate (at either or both termini) in at least one of -P(0)(0H)0-, -0-, -S-, -N(R')-, -C(O)-, -C(0)0-, -OC(O)-, -0C(0)0-, -C(NOR')-, C(0)N(R')-, -C(0)N(R)C(0)-, -C(0)N(R)C(0)N(R , -N(R)C(0)-, -N(R)C(0)N(R)-, - N(R)C(0)0-, -OC(0)N(R)-, -C(NR)-, -N(R')C(NR')-, -C(NR')N(R)-, -N(R')C( R)N(R , -S(0)

2- -OS(O)-, -S(0)0- -S(O)-, -OS(0)₂- , -S(0)₂0-, -N(R)S(0)2-, -S(0) 2N(R)-, -N(R')S(0)-, -

-N(R)S(0)₂N(R')-, -N(R)S(0)N(R)-, C1-C12 carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene or any combination thereof, wherein R is H or C1-C6 alkyl, wherein the interrupting and the one or both terminating groups may be the same or different. In some embodiments, the alkylene chain may comprise 1-30 alkylene units, 1-25 alkylene units, 1-20 alkylene units, 1-15 alkylene units, 1-10 alkylene units, or 1-5 alkylene units.

Linkers may be installed by methods such as illustrated in Figure 6.

The ligase ligand is a functional moiety that binds a ligase, such as E3 ubiquitin ligase. The type of ligase ligand can determine rate and efficiency of protein degradation. In some embodiments, the ligase ligand is an E3 ligase ligand that binds cereblon. Exemplary ligase ligand that binds cereblon include, without limitation, the following structures:

As shown in Figure 7, the E3 ligase ligand may comprise a thalidomide, lenalidomide, or avadomide moiety.

In some embodiments, the ligase ligand binds a Von Hippel-Lindau (VHL) tumor suppressor. Representative examples of ligase ligands that bind VHL are as follows:

wherein X is a bond, N, 0 or C. As shown in Figure 7, the ligase ligand may comprise a VH032 moiety.

An exemplary NEK2 PROTAC, which is used in the Examples that follow, is

The compound comprises a Ne-1 moiety as the NEK2 binding ligand, a thalidomide moiety as the ligase ligand, and linker region having ethylene glycol units that are used to spacably connect the Ne-1 and thalidomide moieties.

The synthesis of NEK2 PROTACs employing Ne-1 as the NEK2 ligand follows a non linear synthetic approach. The synthesis is divided into three parts: (1) NEK2 binding ligand synthesis (Ne-1), (2) linker synthesis (e.g., PEG or triazole-based installation), and (3) ligase ligand synthesis (e.g., thalidomide, lenalidomide, avadomide, or VH 032).

As illustrated in Figure 6, the synthesis of the NEK2 ligand intermediate, INTI -Ne-1, may be synthesized as previously disclosed (Wang J, et al. Targeting NEK2 attenuates glioblastoma growth and radioresistance by destabilizing histone methyltransferase EZH2. J Clin Invest. 2017; 127(8):3075-89). INT2-Ne-1, may be coupled to the linker and E3 ligase ligand, will be synthesized using ethyl lH-pyrazole-l-acetate-3-boronic ester via a Suzuki cross coupling reaction using Suzuki conditions as previously described (id.). The carboxylic ester may be cleaved via acid hydrolysis to generate the corresponding acid intermediate, INT2-Ne-1. Intermediate INT2-Ne-1 may serve as the NEK2 binding ligand that will be coupled to various PEG and triazolebased linkers and lifase ligands such as thalidomide (CRBN), lenalidomide (CRBN), avadomide (CRBN), and VH 032 (VHL). INT2-Ne-1 may also be coupled to propargylamine and 2-azidoethanamine to form the triazole linker on the NEK2 end of the PROTAC.

Linkers based on PEG may be synthesized from commercially available sources, with an aliphatic amine on one end and an aldehyde or amine on the other to attach Ne-1 to the ligase ligand (Figure 6). PEG unit length may vary, e.g., from 1 to 5 units, to allow for protein degradation depending on the selection of ligase ligand and NEK2 binding ligands utilized. Linkers based on triazole may be synthesized from the same PEG starting materials and affixed with an azide or acetylene on one end to ‘click’ the NEK2 ligand to the ligase ligand. Using various linker-lengths with different compositions permits refinement of the NEK2 PROTAC for optimal protein degradation.

Ligase ligands may be prepared from known procedures or purchased commercially, as necessary. Nucleophilic substitution or reductive amination reactions may be used to attach the ligase ligand to the linker. Depending on the linker, the ligase ligand/linker may be attached to fNT2-Ne-l through EDC coupling or click chemistry. Figure 7 illustrates several different ligase ligands and the position of potential attachment to the linker region.

The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more NEK2 PROTAC S as described herein and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to 100 mg/kg body weight (preferably about 0.5 to 20 mg/kg body weight, more preferably about 0.1 to 10 mg/kg body weight). In some embodiments, after the pharmaceutical composition is administered to a patient (e.g., after about 1, 2, 3, 4, 5, or 6 hours post administration), the concentration of the compound at the site of action is about 2 to 10 mM.

In some embodiments, the pharmaceutical composition may further comprise a bioactive agent. The term “bioactive agent” is used to describe an agent, other than a NEK2 PROTAC, which is used in combination with the NEK2 PROTAC as an agent with biological activity to assist in effecting an intended therapy, inhibition and/or prevention/prophylaxis for which the present compounds are used. Exemplary bioactive agents include anti-cancer agents. An "anti- cancer agent" means a compound or composition that can be combined with a NEK2 PROTAC to treat cancer, inhibit the growth or proliferation of a cancer cell, or kill a cancer cell. In some embodiments, the EK2 PROTAC may sensitize a subject to the anti-cancer agent. Exemplary anti-cancer agents include, without limitation, doxorubicin or vincristine.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents.

Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing.

Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route. For example, the pharmaceutical composition may be administered via oral, intravenous, intramuscular, subcutaneous, topical, and pulmonary route. Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders and granules.

The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.

Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

The formulations may be presented in unit-dose or multi-dose containers.

The compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form, which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures. The compounds for use according to the methods of disclosed herein may be administered as a single compound or a combination of compounds. For example, a compound that anti-cancer activity may be administered as a single compound or in combination with another compound that promotes also promotes anti-cancer activity or that has a different pharmacological activity. As indicated above, pharmaceutically acceptable salts of the compounds are contemplated and also may be utilized in the disclosed methods. The term “pharmaceutically acceptable salt” as used herein, refers to salts of the compounds that are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases.

The particular counter-ion forming a part of any salt of a compound disclosed herein is may not be critical to the activity of the compound, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirably solubility or toxicity.

Pharmaceutically acceptable esters and amides of the compounds can also be employed in the compositions and methods disclosed herein. Examples of suitable esters include alkyl, aryl, and aralkyl esters, such as methyl esters, ethyl esters, propyl esters, dodecyl esters, benzyl esters, and the like. Examples of suitable amides include unsubstituted amides, monosub stituted amides, and disubstituted amides, such as methyl amide, dimethyl amide, methyl ethyl amide, and the like.

In addition, the methods disclosed herein may be practiced using solvate forms of the compounds or salts, esters, and/or amides, thereof. Solvate forms may include ethanol solvates, hydrates, and the like.

An aspect of the technology provides for a method for treating of subject in need of any of the NEK2 PROTACs described herein. Suitably, method may comprise administering an effective amount of the NEK2 PROTAC to the subject. As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration. As used herein, a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal, in need of treatment. A “subject in need of treatment” may include a subject having a disease, disorder, or condition that is responsive to therapy with the NEK2 PROTACs disclosed herein, either alone or in combination with another bioactive agent. In some embodiments, a subject in need of treatment may include a subject in need of treatment for a cancer.

In other embodiments, a subject in need of a treatment may include a subject in need sensitization to a bioactive agent, such as anti-cancer agent. The terms "sensitize" and "sensitizing" refer to making, through the administration of a first agent (e.g. , a NEK2 PROTACT of the invention), a subject or a cell more susceptible, or more responsive, to the biological effects (e.g. , promotion or retardation of an aspect of cellular function including, but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis) of a second agent (e.g., the bioactive agent). The sensitizing effect of a first agent on a target cell can be measured as the difference in the intended biological effect (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed upon the administration of a second agent with and without administration of the first agent. The response of the sensitized subject or cell can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% over the response in the absence of the first agent.

As used herein the term “effective amount” refers to the amount or dose of the compound, such as upon single or multiple dose administration to the subject, which provides the desired effect. For example, with respect to the treatment of a cancer, an effective amount will refer to the amount of a therapeutic agent that decreases the rate of tumor growth, decreases tumor mass, decreases the number of metastases, increases time to tumor progression, or increases survival time by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. With respect to sensitization, an effective amount will refer to the amount of a therapeutic agent that results in sensitization of the subject or cell as described above.

An effective amount can be determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

In some embodiments, the EK2 PROTACs described herein may be administered with one or more bioactive agents, including anti-cancer agents. Suitably, the NEK2 PROTAC may be administered before, during, or after administration of the bioactive agent. The NEK2 PROTAC may sensitize the subject to the bioactive agent. This may allow for improved efficacy of the bioactive agent, reduction in an effective amount of bioactive agent needed to achieve a desired effect, or reduce the duration of treatment with the bioactive agent. In some embodiments, the bioactive agent is an anti-cancer agent, such as doxorubicin or vincristine.

As used herein, "cancer" refers to the pathological process that results in the formation and growth of a cancerous or malignant neoplasm, i.e., abnormal tissue that grows by cellular proliferation, often more rapidly than normal and continues to grow after the stimuli that initiated the new growth cease. Exemplary cancers that may be treated by the NEK2 PROTACs described herein may include diffuse large B-cell lymphoma (DLBCL), including AYA DLBCL or ABC DLBCL.

DLBCL is the most common type of non-Hodgkin lymphoma, both worldwide and in the United States. At diagnosis, about half of all patients present with stage I-II disease, whereas the other half present with stage III-IV disease. DLBCL can be further classified by its cell-of-origin, for example germinal center B-cell (GCB, 40-50%) versus activated B-cell like (ABC) (50- 60%). Typically, DLBCL is treated with RCHOP chemotherapy regimens, which can promote long-term, disease-free survival in -60% of patients. Relapsed or refractory patients, however, have poor survival with only -10% achieving curative disease. At the time of diagnosis, patients with GCB neoplasms have higher overall survival than those with ABC tumors. NEK2 is upregulated in both follicular lymphoma (FL) and DLBCL). Of note, when aberrantly expressed in FL, NEK2 drives the progression and transformation into DLBCL, a more aggressive form of lymphoma. Given that NEK2 facilitates centrosome separation at the G2/M stage of the cell cycle, the elevated presence of NEK2 is linked to an increase in chromosome abnormalities, drug resistance, AKT activity, and cell division. A characteristic shared between multiple types of lymphomas is aneuploidy and chromosomal rearrangements. Because of the ability to promote chromosomal stress, NEK2 activity may contribute to the defining features of a lymphoma, enabling disease progression. A large percentage of DLBCL undergo a PTEN knockout rendering the AKT pathway constitutively active. High NEK2 protein levels compromise the protein phosphatase 1 (PP1) regulatory mechanism of AKT, which is independent of PTEN signaling. Therefore, eliminating NEK2 protein levels with an NEK2 PROTAC is a method to reinstate AKT regulation by restoring endogenous phosphatase activity.

DLBCL is classified into two main subtypes based on gene expression patterns resembling the normal counterparts, the germinal center B-cell-like and the activated B-cell- like^9,10. Recent genomic investigations expanded these subtypes into additional molecular groups, further illustrating the complexity of this hematological malignancy^11,12. While these comprehensive analyses characterize the molecules and pathways exploited by this aggressive cancer, age is not often considered beyond exclusion of pediatric cases (NCI-defined age cut-off of <15 years). Studies examining patients that span the pediatric and AYA age groups show, in contrast to adult DLBCL where the germinal center subtype accounts for half of the cases, DLBCL from young patients almost exclusively display a germinal center B-cell-like profile^4,5. This consistent genetic signature indicates AYA DLBCL are not only more homogeneous, but may also exhibit a higher dependence on germinal center B-cell signaling pathways. A notable marker of germinal center B-cell-like DLBCL and prognostically significant oncogene, BCL2, increases with age¹³. The lower expression of this prominent pro-survival factor despite the distinct prevalence of the germinal center subtype in AYA DLBCL suggests alternative oncogenic pathways may promote DLBCL in younger patients.

NEK2 overexpression stimulates germinal center formation and subsequent growth of these B-cell maturation sites³. This active involvement in germinal center programming critical for early B-cell development points towards a role for NEK2 in germinal center-derived lymphomas. NEK2 is a cell cycle associated kinase that regulates AKT pro-survival and proliferation signaling and maintains proper chromosome separation by phosphorylating and localizing key substrates to the centrosome. The Examples demonstrate a strong association of NEK2-directed pathways and AYA DLBCL as well as an induced sensitization to existing chemotherapy following treatment with NEK2 inhibitors. As a result, NEK2 PROTACs increase the efficacy of standard chemotherapy which allows for reducing both the amount and duration of existing treatment. Reducing the dose and frequency of anti-cancer agent used to treat young DLBCL patients would attenuate dose-dependent neuro- and cardio-toxicities and lower the risk of secondary malignancies.

Another aspect of the technology provides for methods for proteolysis of a NEK2 protein. The method may comprise contacting the NEK2 protein with the proteolysis targeting chimera according to the present disclosure. The method may be performed in vivo , in vitro, or ex vivo. In some embodiments, the contacting step is performed within a cell. In some embodiments, the cell is a cancer cell, such as a DLBCL cell including an ABC DLBCL cell or a AYA DLBCL cell.

Another aspect of the technology provides for methods for inhibiting the growth or proliferation of a cell or for killing the cell. The method may comprise contacting the cell with an effective amount of the proteolysis targeting chimera according to the present disclosure to inhibit growth or proliferation or kill the cell. The method may be performed in vivo, in vitro, or ex vivo. In some embodiments, the NEK2 PROTAC contacts EK2 within a cell. In some embodiments, the cell is a cancer cell, such as a DLBCL cell including an ABC DLBCL cell or a AYA DLBCL cell.

Another aspect of the technology provides for methods for sensitizing a cell to a bioactive agent, such as an anti-cancer agent. The method may comprise contacting the cell with an effective amount of the proteolysis targeting chimera according to the present disclosure to sensitize the cell. The method may further comprises contacting the cell with an effective amount of the effective amount of the bioactive or anti-cancer agent. The method may be performed in vivo, in vitro, or ex vivo. In some embodiments, the NEK2 PROTAC contacts EK2 within a cell. In some embodiments, the cell is a cancer cell, such as a DLBCL cell including an ABC DLBCL cell or a AYA DLBCL cell. Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more ” For example, “a molecule” should be interpreted to mean “one or more molecules.”

As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

EXAMPLES

AYA DLBCL express NEK2-dependent genomic and proteomic profiles. Gene expression data from patient-derived DLBCL indicate that, compared to adult tumors, AYA tumors express genes in oncogenic signaling regulated by NEK2, namely the MAPK/AKT/STAT pathways (Fig. 8 (panel A)) and more frequently express NEK2 protein (Fig. 8 (panel B)). Of note, the overall gene signature clustered most of the AYA DLBCL samples together. Tandem mass tag - mass spectrometry (TMT-MS) profding of protein expression in AYA and adult DLBCL cells matched for the germinal center B-cell subtype and female sex confirmed this pro-proliferation/survival phenotype marked by up-regulation of downstream NEK2 substrates, which further supports targeting NEK2 to inhibit tumor growth (Fig. 9 (panel A) and 9 (panel B)). As evidence for the sensitivity of this advanced proteomic approach, we were able to detect age related differences in female-specific pathways (Fig. 9 (panel C)). Genomic and proteomic profiles were determined from the methods described in Storey, AJ etal. Mol. Omics, 2020,16, 316-326.

Inhibiting NEK2 activity induces a robust response on cell cycle and centrosome related substrates. Thus far, we identified differences in protein levels of positive and negative cell cycle regulators and centrosome related proteins, some of which are known NEK2 substrates (NDC80, CROCC, MAP4K1) between the AYA and adult cell lines supporting a pro-mitotic state of the AYA cells (Fig. 9 (panel B)). We then performed a similar proteomic study for total protein assessment, and expanded this analysis to include phospho-proteomics for detecting subsequent effects of inhibiting NEK2 phosphorylation activity. AYA cells treated with the NEK2 inhibitor displayed significant differences in total protein expression (Fig. 10 (panel A)), again consisting of key cell cycle and centrosome related proteins (Fig. 10 (panel B)). Furthermore, the loss of NEK2 catalytic function resulted in a distinct, altered phosphorylation profile with changes in phosphorylation status of NEK2 substrates including MAP2K2 and MAP4K4 (Fig. 10 (panel C)). As expected for a serine/threonine kinase, phosphorylation at these amino acid sites was among those with the highest probability for differences (Fig. 10 (panel D)). The protein assessment were determined from the methods described in Storey, AJ el al. Mol. Omics, 2020,16, 316-326.

NEK2 inhibition sensitizes AYA DLBCL to chemotherapy. In a standard MTS-based cytotoxicity assay, we demonstrate that 96 h co-treatment of our NEK2 kinase inhibitor with either doxorubicin or vincristine selectively sensitized AYA DLBCL cells to non-cytotoxic doses of these two mainstay chemo-agents (Fig. 11 (panel A)) compared to adult DLBCL (Fig. 11 (panel B)) or benign B-cells (Fig. 11 (panel C)). For doxorubicin the effective dose was 16-18 fold lower than the growth inhibitory concentration and 5-20 fold lower for vincristine (Fig. 11 (panel D)). While other AYA cells responded in a similar manner, here, we show SUDHL5 cells from the proteomic studies and one other AYA cell line, U2932, that is also of the recently defined molecular subtype A53¹², to illustrate targeting NEK2 may be effective in treating DLBCL driven in part by aneuploidy, another hallmark of deregulated NEK2¹⁴. Of note, the remarkable potentiation of vincristine indicates a strong synergy between this non-specific, cell cycle drug and an anti-NEK2 strategy (Fig. 11 (panel A), grey bars). This data supports the rationale to validate our bifunctional NEK2 proteolysis-targeting chimera (PROTAC) in enhancing this synergy by limiting additional cancer-enabling, non-catalytic functions of NEK2.

PROTAC strategy effectively targets downstream NEK2 activity. Due to the dual functions of EK2 as both a kinase and scaffold protein, only inhibiting the catalytic activity may not eliminate the oncogenic role of NEK2. Most kinase inhibitors target the ATP binding pocket to block the catalytic transfer of phosphate to substrates¹⁵. These inhibitors are successful in treating several cancer types, but are 1) inefficient at inhibiting kinases with cancer-promoting activity beyond catalysis, such as NEK2, and 2) susceptible to inactivation from mutations to the kinase, which limit their sustained use. As an alternative strategy to address both of these issues, we designed and synthesized bifunctional inhibitors capable of degrading NEK2 using PROTAC technology. With our selective NEK2 inhibitor acting as a ligand to localize the PROTAC to NEK2, an adjoining E3 ligase-binding moiety will induce degradation of NEK2 and inhibit all NEK2 activities. The complete removal of NEK2 from tumor cells will restrict the accumulation of potential NEK2 mutants that would present a future mechanism of resistance. A lead PROTAC candidate consists of our NEK2 inhibitor as the NEK2 ligand, a PEG-based linker, and an E3 ligase ligand (Fig. 5 (panel B)) and triggers a decrease in NEK2 protein levels in AYA DLBCL cells (Fig. 12 (panel A)). Compared to our inhibitor, this NEK2 PROTAC altered the expression and phosphorylation status of an additional 131 and 147 NEK2-associated proteins, respectively, indicating degradation is more effective at inhibiting NEK2 catalytic activity as well as repressing other NEK2 oncogenic functions (Fig. 12 (panel B) and 12 (panel C)). Accordingly, data generated using the PROTACs are expected to provide functional and mechanistic insights into the oncogenic role of NEK2 as well as a new therapeutic approach in AYA DLBCL.

REFERENCES

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Claims

We claim:

1 A proteolysis targeting chimera of formula LL-L-NBL, wherein NBL is a EK2 binding ligand, LL is a ligase ligand, and L is a linker-region to link the NEK2 ligand to the ligase ligand.

2 The proteolysis targeting chimera of claim 1, wherein the NEK2 binding ligand is a Ne-1 moiety.

3. The proteolysis targeting chimera of claim 2, wherein the ligase ligand is an ubiquitin ligase ligand.

4. The proteolysis targeting chimera of claim 3, wherein the ubiquitin ligase ligand comprises a thalidomide, lenalidomide, avadomide, or VH 032 moiety.

5. The proteolysis targeting chimera of claim 2, wherein the linker region comprises 1-5 ethylene glycol units.

6 A proteolysis targeting chimera of formula

7. A pharmaceutical composition comprising an effective amount of the proteolysis targeting chimera according to any one of claims 1-6 and a pharmaceutically acceptable excipient, carrier, or diluent, and optionally further comprising an anti-cancer agent.

8 The pharmaceutical composition of claim 7, wherein composition comprises the anti cancer agent.

9. The pharmaceutical composition of claim 8, wherein the anti-cancer agent is doxorubicin or vincristine.

10. A method for treating of subject in need of a proteolysis targeting chimera comprising administering an effective amount of the proteolysis targeting chimera according to any one of claims 1-6.

11. The method of claim 10, wherein the subject is in need of the proteolysis targeting chimera for treating a cancer.

12. The method of claim 11, wherein the cancer is a DLBCL.

13. The method of claim 12, wherein the cancer is an AYA DLBCL.

14. The method of claim 12, wherein the cancer is an ABC DLBCL.

15. The method of claim 10, wherein the subject is in need of the proteolysis targeting chimera comprising for sensitizing the subject to an anti-cancer agent.

16. The method of claim 15 further comprising administering an effective amount of the anti cancer agent.

17. The method of claim 16, wherein the anti-cancer agent is doxorubicin or vincristine.

18. The method of any one of claims 15-17, the subject is in need of the proteolysis targeting chimera for treating a cancer.

19. The method of claim 18, wherein the cancer is a DLBCL.

20. The method of claim 19, wherein the cancer is an AYA DLBCL.

21. The method of claim 19, wherein the cancer is an ABC DLBCL.

22. A method for proteolysis of a NEK2 protein comprising contacting the NEK2 protein with the proteolysis targeting chimera according to any one of claims 1-6.

23. The method of claim 22, wherein the NEK2 protein is contacted with the proteolysis targeting chimera in a cell.

24. The method of claim 23, wherein the cell is a cancer cell.

25. The method of claim 24, wherein the cancer is a DLBCL.

26. The method of claim 25, wherein the cancer is an AYA DLBCL.

27. The method of claim 25, wherein the cancer is an ABC DLBCL.

28. A method for inhibiting the growth or proliferation of a cell or for killing the cell, the method comprising contacting the cell with an effective amount of the proteolysis targeting chimera according to any one of claims 1-6.

29. The method of claim 28, wherein the cell is a cancer cell.

30. The method of claim 29, wherein the cancer is a DLBCL.

31. The method of claim 30, wherein the cancer is an AYA DLBCL.

32. The method of claim 30, wherein the cancer is an ABC DLBCL.

33. A method for sensitizing a cell to an anti-cancer agent, the method comprising contacting the cell with an effective amount of the proteolysis targeting chimera according to any one of claims 1-6.

34. The method of claim 33, wherein the cell is a cancer cell.

35. The method of claim 34, wherein the cancer is a DLBCL.

36. The method of claim 35, wherein the cancer is an AYA DLBCL.

37. The method of claim 35, wherein the cancer is an ABC DLBCL.

38. The method of any one of claims 33-37, further comprising contacting the cell with an effective amount of the effective amount of the anti-cancer agent.

39. The method of claim 38, wherein the anti-cancer agent is doxorubicin or vincristine.