Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias.

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Catalytic Degraders Effectively Address Kinase Site Mutations in EML4-ALK Oncogenic Fusions.

Heterobifunctional degraders, known as proteolysis targeting chimeras (PROTACs), theoretically possess a catalytic mode-of-action, yet few studies have either confirmed or exploited this potential advantage of event-driven pharmacology. Degraders of oncogenic EML4-ALK fusions were developed by conjugating ALK inhibitors to cereblon ligands. Simultaneous optimization of pharmacology and compound properties using ternary complex modeling and physicochemical considerations yielded multiple catalytic degraders that were more resilient to clinically relevant ATP-binding site mutations than kinase inhibitor drugs. Our strategy culminated in the design of the orally bioavailable derivative CPD-1224 that avoided hemolysis (a feature of detergent-like PROTACs), degraded the otherwise recalcitrant mutant L1196M/G1202R in vivo, and commensurately slowed tumor growth, while the third generation ALK inhibitor drug lorlatinib had no effect. These results validate our original therapeutic hypothesis by exemplifying opportunities for catalytic degraders to proactively address binding site resistant mutations in cancer.

CDK7 controls E2F- and MYC-driven proliferative and metabolic vulnerabilities in multiple myeloma.

Therapeutic targeting of CDK7 has proven beneficial in preclinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in cells from patients with multiple myeloma (MM); its selective targeting counteracts E2F activity via perturbation of the cyclin-dependent kinases/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small-molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression, increasing survival in several mouse models of MM including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use.

Structure-Based Design of Y-Shaped Covalent TEAD Inhibitors.

Transcriptional enhanced associate domain (TEAD) proteins together with their transcriptional coactivator yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) are important transcription factors and cofactors that regulate gene expression in the Hippo pathway. In mammals, the TEAD families have four homologues: TEAD1 (TEF-1), TEAD2 (TEF-4), TEAD3 (TEF-5), and TEAD4 (TEF-3). Aberrant expression and hyperactivation of TEAD/YAP signaling have been implicated in a variety of malignancies. Recently, TEADs were recognized as being palmitoylated in cells, and the lipophilic palmitate pocket has been successfully targeted by both covalent and noncovalent ligands. In this report, we present the medicinal chemistry effort to develop MYF-03-176 (compound 22) as a selective, cysteine-covalent TEAD inhibitor. MYF-03-176 (compound 22) significantly inhibits TEAD-regulated gene expression and proliferation of the cell lines with TEAD dependence including those derived from mesothelioma and liposarcoma.

Human Polo-like Kinase Inhibitors as Antiplasmodials.

Protein kinases have proven to be a very productive class of therapeutic targets, and over 90 inhibitors are currently in clinical use primarily for the treatment of cancer. Repurposing these inhibitors as antimalarials could provide an accelerated path to drug development. In this study, we identified BI-2536, a known potent human polo-like kinase 1 inhibitor, with low nanomolar antiplasmodial activity. Screening of additional PLK1 inhibitors revealed further antiplasmodial candidates despite the lack of an obvious orthologue of PLKs in Plasmodium. A subset of these inhibitors was profiled for their in vitro killing profile, and commonalities between the killing rate and inhibition of nuclear replication were noted. A kinase panel screen identified PfNEK3 as a shared target of these PLK1 inhibitors; however, phosphoproteome analysis confirmed distinct signaling pathways were disrupted by two structurally distinct inhibitors, suggesting PfNEK3 may not be the sole target. Genomic analysis of BI-2536-resistant parasites revealed mutations in genes associated with the starvation-induced stress response, suggesting BI-2536 may also inhibit an aminoacyl-tRNA synthetase.

Covalent disruptor of YAP-TEAD association suppresses defective Hippo signaling.

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

A Novel HER2-Selective Kinase Inhibitor Is Effective in HER2 Mutant and Amplified Non-Small Cell Lung Cancer.

In-frame insertions in exon 20 of HER2 are the most common HER2 mutations in patients with non-small cell lung cancer (NSCLC), a disease in which approved EGFR/HER2 tyrosine kinase inhibitors (TKI) display poor efficiency and undesirable side effects due to their strong inhibition of wild-type (WT) EGFR. Here, we report a HER2-selective covalent TKI, JBJ-08-178-01, that targets multiple HER2 activating mutations, including exon 20 insertions as well as amplification. JBJ-08-178-01 displayed strong selectivity toward HER2 mutants over WT EGFR compared with other EGFR/HER2 TKIs. Determination of the crystal structure of HER2 in complex with JBJ-08-178-01 suggests that an interaction between the inhibitor and Ser783 may be responsible for HER2 selectivity. The compound showed strong antitumoral activity in HER2-mutant or amplified cancers in vitro and in vivo. Treatment with JBJ-08-178-01 also led to a reduction in total HER2 by promoting proteasomal degradation of the receptor. Taken together, the dual activity of JBJ-08-178-01 as a selective inhibitor and destabilizer of HER2 represents a combination that may lead to better efficacy and tolerance in patients with NSCLC harboring HER2 genetic alterations or amplification. SIGNIFICANCE: This study describes unique mechanisms of action of a new mutant-selective HER2 kinase inhibitor that reduces both kinase activity and protein levels of HER2 in lung cancer.

Novel Macrocyclic Peptidomimetics Targeting the Polo-Box Domain of Polo-Like Kinase 1.

The polo-box domain (PBD) of Plk1 is a promising target for cancer therapeutics. We designed and synthesized novel phosphorylated macrocyclic peptidomimetics targeting PBD based on acyclic phosphopeptide PMQSpTPL. The inhibitory activities of 16e on Plk1-PBD is >30-fold higher than those of PMQSpTPL. Both 16a and 16e possess excellent selectivity for Plk1-PBD over Plk2/3-PBD. Analysis of the cocrystal structure of Plk1-PBD in complex with 16a reveals that the 3-(trifluoromethyl)benzoyl group in 16a interacts with Arg516 through a π-stacking interaction. This π-stacking interaction, which has not been reported previously, provides insight into the design of novel and potent Plk1-PBD inhibitors. Furthermore, 16h, a PEGlyated macrocyclic phosphopeptide derivative, induces Plk1 delocalization and mitotic failure in HeLa cells. Also, the number of phospho-H3-positive cells in a zebrafish embryo increases in proportion to the amount of 16a. Collectively, the novel macrocyclic peptidomimetics should serve as valuable templates for the design of potent and novel Plk1-PBD inhibitors.

Targeting transcription cycles in cancer.

Accurate control of gene expression is essential for normal development and dysregulation of transcription underpins cancer onset and progression. Similar to cell cycle regulation, RNA polymerase II-driven transcription can be considered as a unidirectional multistep cycle, with thousands of unique transcription cycles occurring in concert within each cell. Each transcription cycle comprises recruitment, initiation, pausing, elongation, termination and recycling stages that are tightly controlled by the coordinated action of transcriptional cyclin-dependent kinases and their cognate cyclins as well as the opposing activity of transcriptional phosphatases. Oncogenic dysregulation of transcription can entail defective control of gene expression, either at select loci or more globally, impacting a large proportion of the genome. The resultant dependency on the core-transcriptional machinery is believed to render 'transcriptionally addicted' cancers sensitive to perturbation of transcription. Based on these findings, small molecules targeting transcriptional cyclin-dependent kinases and associated proteins hold promise for the treatment of cancer. Here, we utilize the transcription cycles concept to explain how dysregulation of these finely tuned gene expression processes may drive tumorigenesis and how therapeutically beneficial responses may arise from global or selective transcriptional perturbation. This conceptual framework helps to explain tumour-selective transcriptional dependencies and facilitates the rational design of combination therapies.

Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma.

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.

Discovery and resistance mechanism of a selective CDK12 degrader.

Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules.

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Synergistic Anti-Tumor Effect of Combining Selective CDK7 and BRD4 Inhibition in Neuroblastoma.

PURPOSE: Cyclin-dependent kinases (CDKs) that have critical roles in RNA polymerase II (Pol II)-mediated gene transcription are emerging as therapeutic targets in cancer. We have previously shown that THZ1, a covalent inhibitor of CDKs 7/12/13, leads to cytotoxicity in MYCN-amplified neuroblastoma through the downregulation of super-enhancer-associated transcriptional upregulation. Here we determined the effects of YKL-5-124, a novel covalent inhibitor with greater selectivity for CDK7 in neuroblastoma cells. EXPERIMENTAL DESIGN: We tested YKL-5-124 in MYCN-amplified and nonamplified neuroblastoma cells individually and in combination with other inhibitors in cell line and animal models. Cell viability, target validation, effects on cell cycle and transcription were analyzed. RESULTS: CDK7 inhibition with YKL-5-124 did not lead to significant cell death, but resulted in aberrant cell cycle progression especially in MYCN-amplified cells. Unlike THZ1, YKL-5-124 had minimal effects on Pol II C-terminal domain phosphorylation, but significantly inhibited that of the CDK1 and CDK2 cell cycle kinases. Combining YKL-5-124 with the BRD4 inhibitor JQ1 resulted in synergistic cytotoxicity. A distinct MYCN-gene expression signature associated with resistance to BRD4 inhibition was suppressed with the combination. The synergy between YKL-5-124 and JQ1 translated into significant tumor regression in cell line and patient-derived xenograft mouse models of neuroblastoma. CONCLUSIONS: The combination of CDK7 and BRD4 inhibition provides a therapeutic option for neuroblastoma and suggests that the addition of YKL-5-124 could improve the therapeutic efficacy of JQ1 and delay resistance to BRD4 inhibition.

Discovery of MFH290: A Potent and Highly Selective Covalent Inhibitor for Cyclin-Dependent Kinase 12/13.

Genetic depletion of cyclin-dependent kinase 12 (CDK12) or selective inhibition of an analog-sensitive CDK12 reduces DNA damage repair gene expression, but selective inhibition of endogenous CDK12 is difficult. Here, we report the development of MFH290, a novel cysteine (Cys)-directed covalent inhibitor of CDK12/13. MFH290 forms a covalent bond with Cys-1039 of CDK12, exhibits excellent kinome selectivity, inhibits the phosphorylation of serine-2 in the C-terminal domain (CTD) of RNA-polymerase II (Pol II), and reduces the expression of key DNA damage repair genes. Importantly, these effects were demonstrated to be CDK12-dependent as mutation of Cys-1039 rendered the kinase refractory to MFH290 and restored Pol II CTD phosphorylation and DNA damage repair gene expression. Consistent with its effect on DNA damage repair gene expression, MFH290 augments the antiproliferative effect of the PARP inhibitor olaparib.

Development of CDK2 and CDK5 Dual Degrader TMX-2172.

Cyclin-dependent kinase 2 (CDK2) is a potential therapeutic target for the treatment of cancer. Development of CDK2 inhibitors has been extremely challenging as its ATP-binding site shares high similarity with CDK1, a related kinase whose inhibition causes toxic effects. Here, we report the development of TMX-2172, a heterobifunctional CDK2 degrader with degradation selectivity for CDK2 and CDK5 over not only CDK1, but transcriptional CDKs (CDK7 and CDK9) and cell cycle CDKs (CDK4 and CDK6) as well. In addition, we demonstrate that antiproliferative activity in ovarian cancer cells (OVCAR8) depends on CDK2 degradation and correlates with high expression of cyclin E1 (CCNE1), which functions as a regulatory subunit of CDK2. Collectively, our work provides evidence that TMX-2172 represents a lead for further development and that CDK2 degradation is a potentially valuable therapeutic strategy in ovarian and other cancers that overexpress CCNE1.

Targeting the PI5P4K Lipid Kinase Family in Cancer Using Covalent Inhibitors.

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/ß/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

CDK7 Inhibition Potentiates Genome Instability Triggering Anti-tumor Immunity in Small Cell Lung Cancer.

Cyclin-dependent kinase 7 (CDK7) is a central regulator of the cell cycle and gene transcription. However, little is known about its impact on genomic instability and cancer immunity. Using a selective CDK7 inhibitor, YKL-5-124, we demonstrated that CDK7 inhibition predominately disrupts cell-cycle progression and induces DNA replication stress and genome instability in small cell lung cancer (SCLC) while simultaneously triggering immune-response signaling. These tumor-intrinsic events provoke a robust immune surveillance program elicited by T cells, which is further enhanced by the addition of immune-checkpoint blockade. Combining YKL-5-124 with anti-PD-1 offers significant survival benefit in multiple highly aggressive murine models of SCLC, providing a rationale for new combination regimens consisting of CDK7 inhibitors and immunotherapies.

A kinase-independent role for CDK8 in BCR-ABL1+ leukemia.

Cyclin-dependent kinases (CDKs) are frequently deregulated in cancer and represent promising drug targets. We provide evidence that CDK8 has a key role in B-ALL. Loss of CDK8 in leukemia mouse models significantly enhances disease latency and prevents disease maintenance. Loss of CDK8 is associated with pronounced transcriptional changes, whereas inhibiting CDK8 kinase activity has minimal effects. Gene set enrichment analysis suggests that the mTOR signaling pathway is deregulated in CDK8-deficient cells and, accordingly, these cells are highly sensitive to mTOR inhibitors. Analysis of large cohorts of human ALL and AML patients reveals a significant correlation between the level of CDK8 and of mTOR pathway members. We have synthesized a small molecule YKL-06-101 that combines mTOR inhibition and degradation of CDK8, and induces cell death in human leukemic cells. We propose that simultaneous CDK8 degradation and mTOR inhibition might represent a potential therapeutic strategy for the treatment of ALL patients.

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation.

Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.

Development of a Selective CDK7 Covalent Inhibitor Reveals Predominant Cell-Cycle Phenotype.

Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G1/S transition and inhibition of E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may be useful for treatment of cancers marked by E2F misregulation.