CDK Inhibitor Research Articles

Quest for discovering novel CDK12 inhibitor.

CDK12 is essential for cellular processes like RNA processing, transcription, and cell cycle regulation, inhibiting cancer cell growth and facilitating macrophage invasion. CDK12 is a significant oncogenic factor in various cancers, including HER2-positive breast cancer, Anaplastic thyroid carcinoma, Hepatocellular carcinoma, prostate cancer, and Ewing sarcoma. It is also regarded as a potential biomarker, emphasizing its broader significance in oncology. Targeting CDK12 offers a promising strategy to develop therapy. Various monoclonal antibodies have drawn wide attention, but they are expensive compared to small-molecule inhibitors, limiting their accessibility and affordability for patients. Consequently, this research aims to identify effective CDK12 inhibitors using comprehensive high-throughput virtual screening. RASPD protocol has been employed to screen three different databases against the target followed by drug-likeness, molecular docking, ADME, toxicity, Consensus molecular docking, MD Simulation, and in-vitro studies MTT assay. The research conducted yielded one compound ZINC11784547 has demonstrated robust binding affinity, favorable ADME features, less toxicity, remarkable stability, and cytotoxic effect. The identified compound holds promise for promoting cancer cell death through CDK12 inhibition.

Cytotoxic effects of the standardized extract from Curcuma aromatica Salisb. rhizomes via induction of mitochondria-mediated caspase-dependent apoptotic pathway and p21-mediated G0/G1 cell cycle arrest on human gastric cancer AGS cells

ABSTRACT Curcuma aromatica Salisb. (C. aromatica) is one of the traditional herbs used to treat microbial infection, skin eruption, coronary heart disease, and other diseases, including cancer. However, the inhibitory effects and underlying mechanisms of action of C. aromatica on gastric cancer cells have not yet been fully elucidated. Our study aimed to examine the possible molecular mechanisms underlying the cytotoxic effects attributed to C. aromatica rhizome standardized extract against gastric cancer cells. The components of two major active compounds in C. aromatica rhizome extract were quantitatively analyzed using a simple and validated HPLC method. Cytotoxicity was determined in different gastric cancer and non-cancer cell lines. The biological activities of the extract targeting apoptosis and cell cycle-related genes on gastric cancer AGS cells were also investigated to elucidate the mechanisms relating to the anti-proliferative effect of C. aromatica rhizomes. The two major active compounds curdione and germacrone, in the C. aromatica extract were standardized to 0.64% and 1.12% w/w, respectively. The standardized extract (CAE) exerted cytotoxic effects on various cancer cells, whereas minimal effects at equivalent doses were noted for normal cells. CAE concentration-dependently suppressed growth of gastric cancer AGS cells via induction of apoptosis. Further studies revealed that CAE treatment disrupted mitochondrial membrane potential (ΔΨm), increased Bax/Bcl-2 ratio, and cytochrome c release, resulting in activation of caspase-9/-3 and subsequent cleavage of PARP. Further, the inhibitory effects of caspase-9/-3 expression by a synthetic pan-caspase inhibitor partially protected cells against apoptosis following CAE treatment. In addition, CAE significantly promoted cell death in AGS cells via an accumulation of cells in the G0/G1 phase. This effect was associated with upregulation of the CDK inhibitor p21 and downregulation of cyclin D1, cyclin E, CDK4, and CDK2 expression. Our data indicated that CAE exerted anti-proliferative activity by activating the mitochondria-mediated caspase-dependent apoptotic pathway and arresting the p21-mediated G0/G1 cell cycle on human gastric cancer AGS cells.

The role of super-enhancers in head and neck cancer and its potential therapeutic targets

Head and neck squamous cell carcinoma (HNSCC) poses a significant global health challenge, with over 660,000 new cases diagnosed and more than 325,000 deaths each year. Despite advances in treatment, long-term survival rates for HNSCC patients remain disappointingly low, underscoring the critical need for innovative therapeutic strategies. This review delves into the role of super-enhancers in HNSCC, highlighting their pivotal function in regulating key oncogenes such as KLF4, FOSL1, and ΔNp63, which are crucial for tumor progression and metastasis. Moreover, it explores how super-enhancers contribute to the maintenance of cancer stem cell characteristics by controlling genes like SOX2, BRD4. thereby promoting self-renewal and pluripotency.The study also underscores the potential of BET bromodomain inhibitors, exemplified by JQ1, and CDK7 inhibitors like THZ1, which demonstrate substantial therapeutic promise by effectively disrupting the function of super-enhancers in HNSCC. Overall, this research provides a comprehensive overview of the importance of super-enhancers in HNSCC .

Sanguinarine identified as a natural dual inhibitor of AURKA and CDK2 through network pharmacology and bioinformatics approaches

Cervical cancer (CA) continues to be a female malignant tumor with limited therapeutic options, resulting in a high mortality rate. Sanguinarine (SANG), a naturally occurring alkaloid, has demonstrated notable efficacy in preclinical treatment of CA. However, the mechanism through which SANG acts against CA is not fully understood. To address this, utilizing nine drug target prediction databases, we have successfully identified 379 potential targets for SANG. Venn diagram analysis compared 2367 CA-related targets from the GeneCards disease database, 2618 CA-closely related targets derived from multiple datasets in GEO through WGCNA analysis, and the 379 potential targets of SANG, resulting in 35 shared targets. Subsequently, by employing PPI network analysis, the Cytohubba plugin, the Human Protein Atlas, TCGA database data, and ROC curve analysis, we have identified AURKA and CDK2 as key targets of SANG in combating CA. Single-gene GSEA results suggest that the overexpression of AURKA and CDK2 is closely correlated with DNA replication, cell cycle progression, and various DNA repair pathways in CA. Molecular docking and molecular simulation dynamics analyses have confirmed the stable binding of both AURKA and CDK2 to SANG. In summary, by integrating diverse methodological approaches, this study discovered that SANG potentially inhibits the malignant features of CA by targeting AURKA and CDK2, thereby regulating DNA replication, cell cycle progression, and multiple DNA repair pathways. This lays a solid foundation for further exploring the pharmacological role of SANG in CA therapy. However, further in-depth in vitro and in vivo experiments are required to corroborate our findings.

Targeting CDK7 enhances the antitumor efficacy of enzalutamide in androgen receptor-positive triple-negative breast cancer by inhibiting c-MYC-mediated tumorigenesis.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Among TNBC subtypes, the luminal androgen receptor (LAR) subtype expresses high levels of androgen receptor (AR) and generally responds poorly to neoadjuvant chemotherapy. AR has been reported as a promising therapeutic target for the LAR TNBC subtype. Here, we evaluated the preclinical antitumor efficacy of enzalutamide, an AR inhibitor, in TNBC. Enzalutamide had moderate anti-proliferation activity against AR-positive (AR+) TNBC cells (IC50 > 15 µM). To enhance its antitumor efficacy, we performed high-throughput kinome siRNA screening and identified the cell cycle pathway as a potential target. Inhibition of cell cycle progression using the CDK7 inhibitor KRLS-017 showed a synergistic anti-proliferation effect with enzalutamide in AR+ LAR MDA-MB-453 and SUM185 TNBC cells. Downstream target analysis revealed that enzalutamide and KRLS-017 combination dramatically reduced c-MYC expression at both mRNA and protein levels. c-MYC knockdown significantly suppressed growth of MDA-MB-453 and SUM185 cells to a degree comparable to that of enzalutamide and KRLS-017 combination treatment, whereas c-MYC overexpression reversed the synergistic effect. An enhancement in inhibition of tumor growth and suppression of c-MYC expression was further confirmed when enzalutamide combined with KRLS-017 in an MDA-MB-453 mouse model. Our study suggests that KRLS-017 enhances the antitumor efficacy of enzalutamide by inhibiting c-MYC-mediated tumorigenesis and presents a potential new approach for treating AR+ LAR TNBC.

Preclinical efficacy of CDK7 inhibitor–based combinations against myeloproliferative neoplasms transformed to AML

AbstractRising blast percentage or secondary acute myeloid leukemia (sAML) transformation in myeloproliferative neoplasms (MPNs) leads to JAK inhibitor (JAKi) therapy resistance and poor survival. Here, we demonstrate that treatment with the CDK7 inhibitor (CDK7i) SY-5609 depletes phenotypically characterized post-MPN sAML stem/progenitor cells. In cultured post-MPN sAML SET2 and HEL as well as patient-derived (PD) post-MPN sAML cells, SY-5609 treatment inhibited growth and induced lethality while sparing normal cells. RNA-sequencing analysis after SY-5609 treatment demonstrated reduced messenger RNA (mRNA) expression of MYC, MYB, CDK4/6, PIM1, and CCND1 but increased mRNA levels of CDKN1A and BCL2L1. Mass spectrometry of SY-5609–treated MPN-sAML cells also demonstrated reduced c-Myc, c-Myb, PIM1, and CDK4/6 but increased p21, caspase 9, and BAD protein levels. CRISPR-mediated CDK7 depletion also reduced the viability of HEL cells. CyTOF analysis of SY-5609–treated PD post-MPN sAML stem/progenitor cells showed reduced c-Myc, CDK6, and PU.1 but increased protein levels of CD11b, p21, and cleaved caspase 3. Cotreatment with SY-5609 and ruxolitinib was synergistically lethal in HEL, SET2, and PD post-MPN sAML cells. A CRISPR screen in SET2 and HEL cells revealed BRD4, CBP, and p300 as codependencies with SY-5609 treatment. Accordingly, cotreatment with SY-5609 and the BETi OTX015 or pelabresib or with the CBP/p300 inhibitor GNE-049 was synergistically lethal in MPN-sAML cells (including those exhibiting TP53 loss). Finally, in the HEL-Luc/GFP xenograft model, compared with each agent alone, cotreatment with SY-5609 and OTX015 reduced post-MPN sAML burden and improved survival without inducing host toxicity. These findings demonstrate promising preclinical activity of the CDK7i-based combinations with BETi or HATi against advanced MPNs, including post-MPN sAML.

LDC000067, a CDK9 inhibitor, unveils promising results in suppressing influenza virus infections in vitro and in vivo.

Influenza virus infections continue to pose a significant threat to public health. Current anti-influenza drugs target viral proteins; however, the emergence of resistant strains has hampered their effectiveness. Fortunately, as with most viruses, influenza virus depends on various host factors during its replication cycle and in pathogenicity. Therefore, the manipulation of key host factors for viral replication to combat influenza has garnered increased attention due to its lesser tendency to induce viral mutation. Cyclin-dependent kinases (CDKs) are a family of protein kinases that regulate various cellular processes, including the cell cycle and transcription. While the specific involvement of CDKs in the transcription of influenza virus genes is less extensively studied than their roles in the cell cycle, some evidence suggests their potential contributions as anti-influenza drugs. Here, we report that LDC000067 (LDC), a highly specific CDK9 inhibitor, not only strongly suppressed influenza virus replication in vitro and in vivo but also emerged as a potential candidate for anti-influenza virus agents. Further investigation revealed that inhibition of CDK9 by LDC treatment and CDK9 silencing disrupts viral RNA transcription and the nuclear import of vRNPs, significantly suppressing influenza virus replication. Mechanistically, we showed that LDC treatment and CDK9 silencing reduce Pol II expressions, a requisite host protein for viral RNA transcription. Altogether, our findings indicate that CDK9 could be a promising target for developing antivirals against influenza virus infections, and LDC, with its strong anti-influenza properties, instills confidence in its potential as an effective anti-influenza agent.

The antitumor peptide M1-20 induced the degradation of CDK1 through CUL4-DDB1-DCAF1-involved ubiquitination.

CDK1 is an oncogenic serine/threonine kinase known to play an important role in the regulation of the cell cycle. FOXM1, as one of the CDK1 substrates, requires binding of CDK1/CCNB1 complex for phosphorylation-dependent recruitment of p300/CBP coactivators to mediate transcriptional activity. Previous studies from our laboratory found that a novel peptide (M1-20) derived from the C-terminus of FOXM1 exhibited potent inhibitory effects for cancer cells. Based on these proofs and to explore the inhibitory mechanism of M1-20, we designed experiments and found that CDK1 served as an important target of M1-20. M1-20 enhanced the ubiquitination and degradation of CDK1 by CUL4-DDB1-DCAF1 complexes through the proteasome pathway. M1-20 could also affect the formation of CDK1/CCNB1 complexes. In addition, compared to RO3306, a CDK1 inhibitor, M1-20 exhibited excellent inhibitory effects in FVB/N MMTV-PyVT murine model of spontaneous breast cancer. These results suggested that M1-20 was a potential CDK1 inhibitor for the treatment of cancer.

Deciphering molecular landscape of breast cancer progression and insights from functional genomics and therapeutic explorations followed by in vitro validation

Breast cancer is caused by aberrant breast cells that proliferate and develop into tumors. Tumors have the potential to spread throughout the body and become lethal if ignored. Metastasis is the process by which invasive tumors move to neighboring lymph nodes or other organs. Metastasis can be lethal and perhaps fatal. The objective of our study was to elucidate the molecular mechanisms underlying the transition of Ductal Carcinoma In Situ (DCIS) to Invasive Ductal Carcinoma (IDC), with a particular focus on hub genes and potential therapeutic agents. Using Weighted Gene Co-expression Network Analysis (WGCNA), we built a comprehensive network combining clinical and phenotypic data from both DCIS and IDC. Modules within this network, correlated with specific phenotypic traits, were identified, and hub genes were identified as critical markers. Receiver Operating Characteristic (ROC) analysis assessed their potential as biomarkers, while survival curve analysis gauged their prognostic value. Furthermore, molecular docking predicted interactions with potential therapeutic agents. Ten hub genes—CDK1, KIF11, NUF2, ASPM, CDCA8, CENPF, DTL, EXO1, KIF2C, and ZWINT—emerged as pivotal fibroblast-specific genes potentially involved in the DCIS to IDC transition. These genes exhibited pronounced positive correlations with key pathways like the cell cycle and DNA repair, Molecular docking revealed Fisetin, an anti-inflammatory compound, effectively binding to both CDK1 and DTL underscoring their role in orchestrating cellular transformation. CDK1 and DTL were selected for molecular docking with CDK1 inhibitors, revealing effective binding of Fisetin, an anti-inflammatory compound, to both. Of the identified hub genes, DTL—an E3 ubiquitin ligase linked to the CRL4 complex—plays a central role in cancer progression, impacting tumor growth, invasion, and metastasis, as well as cell cycle regulation and epithelial-mesenchymal transition (EMT). CDK1, another hub gene, is pivotal in cell cycle progression and associated with various biological processes. In conclusion, our study offers insights into the complex mechanisms driving the transition from DCIS to IDC. It underscores the importance of hub genes and their potential interactions with therapeutic agents, particularly Fisetin. By shedding light on the interplay between CDK1 and DTL expression, our findings contribute to understanding the regulatory landscape of invasive ductal carcinoma and pave the way for future investigations and novel therapeutic avenues.

Comprehensive multi-omics analysis elucidates colchicine-induced toxicity mechanisms and unveils the therapeutic potential of MLN4924 and kinase inhibitors.

Colchicine is a widely prescribed anti-inflammatory drug for the treatment of gout, familial Mediterranean fever and pericarditis, but its narrow therapeutic window presents a significant risk of severe toxicity. Despite its clinical relevance, the molecular mechanisms underlying colchicine's pharmacological effects and associated toxicity and explored potential therapeutic interventions to mitigate its adverse effects. We showed the colchicine's impact on cellular morphology in human umbilical vein endothelial cells (HUVEC) and HeLa cells including cell rounding and detachment following 24 h of exposure that revealed pronounced cytotoxic effects. We then established a large-scale screening model to identify small molecules capable of reversing colchicine-induced cellular toxicity, and identified MLN4924, an inhibitor of the Cullin-RING E3 ligase (CRL) system, as a promising candidate for mitigating colchicine-induced cellular injury. Through a comprehensive multi-omics approach including transcriptomics, proteomics, phosphoproteomics and ubiquitinomics, we systematically characterized the molecular perturbations caused by colchicine and delineated the protective mechanisms of MLN4924. We found that MLN4924 exerted its protective effects by modulating critical cellular pathways, specifically preventing the dysregulation of cell cycle progression, mitotic disruption and microtubule destabilization triggered by colchicine. Furthermore, proteomic and phosphoproteomic analyses revealed significant alterations in kinase signaling networks, with combined inhibition of CDK1 and PAK1 emerging as an effective strategy to counteract colchicine-induced cellular dysfunction. These results not only provide a detailed molecular characterization of colchicine toxicity but also identify key therapeutic targets, laying the groundwork for the development of targeted interventions to mitigate colchicine-induced adverse effects in clinical practice.

Therapeutic benefits of maintaining CDK4/6 inhibitors and incorporating CDK2 inhibitors beyond progression in breast cancer.

The combination of CDK4/6 inhibitors (CDK4/6i) and endocrine therapy has revolutionized treatment for hormone receptor-positive (HR+) metastatic breast cancer. However, the emergence of resistance in most patients often leads to treatment discontinuation with no consensus on effective second-line therapies. The therapeutic benefits of maintaining CDK4/6i or incorporating CDK2 inhibitors (CDK2i) after disease progression remain unclear. Here, we demonstrate that sustained CDK4/6i therapy, either alone or combined with CDK2i, significantly suppresses the growth of drug-resistant HR + breast cancer. Continued CDK4/6i treatment induces a non-canonical pathway for retinoblastoma protein (Rb) inactivation via post-translational degradation, resulting in diminished E2F activity and delayed G1 progression. Importantly, our data highlight that CDK2i should be combined with CDK4/6i to effectively suppress CDK2 activity and overcome resistance. We also identify cyclin E overexpression as a key driver of resistance to CDK4/6 and CDK2 inhibition. These findings provide crucial insights into overcoming resistance in HR + breast cancer, supporting the continued use of CDK4/6i and the strategic incorporation of CDK2i to improve therapeutic outcomes.

In vivo evaluation of novel synthetic pyrazolones as CDK9 inhibitors with enhanced pharmacokinetic properties.

Aim: The structural optimization of our recently reported CDK9 inhibitor to furnish novel aminopyrazolones and methylpyrazolones with improved pharmacokinetics.Materials & methods: The synthesis of the targeted compounds was accomplished via conventional, grinding and microwave-assisted processes. The cytotoxicity of them was assayed against three carcinomas.Results: Analogs 2, 4 and 6 showed significant cytotoxicity and selectivity toward all tested cells. They also displayed potent CDK9 inhibition. Compound 6 arrested MCF-7 cycle at G2/M phase by stimulating the apoptotic pathway. The in vivo biodistribution of radiolabeled compound 6 displayed a potent targeting capability of 131I in solid tumors.Conclusion: Entity 6 is a potent CDK9 inhibitor where 131I-compound 6 can be used as a significant radiopharmaceutical imaging tool for tumors.

EXTH-72. INTEGRATED MULTIOMIC ANALYSIS OF ISOGENIC EGFRVIII MODELS OF GBM REVEALS EXPLOITABLE THERAPEUTIC VULNERABILITIES

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor with an abysmal 15-month median survival. Therefore, novel therapeutic interventions are urgently needed. EGFR is a receptor tyrosine kinase that is mutated in over 50% of GBM and is a logical target for precision oncology approaches. While aberrant EGFR signaling has been successfully targeted in other cancers, early attempts to target EGFR in GBM clinical trials have not been successful. Since these early trials, several studies have revealed that GBM EGFR biology is unique and cannot be generalized from other EGFR-driven neoplasms. To better understand EGFR biology in a GBM-specific context, we have characterized the GBM transcriptome with RNAseq, the epigenome with CUT&RUN, and the kinase proteome with Multiplexed inhibitor beads with Mass Spectrometry (MIB-MS), collectively referred to as ‘multiomics’, after modeling EGFR resistance. An isogenic mouse astrocyte (mAc) model of GBM was genetically engineered to overexpress EGFRvIII (CEv3), the most common EGFR mutation in GBM. Expression of EGFRvIII induces a unique multiomic profile that mediates many hallmark cancer phenotypes including proliferation and stemness. Chronic EGFR resistance was modeled both in vitro and in vivo through continuous exposure to erlotinib or gefitinib, EGFR tyrosine kinase inhibitors (TKI). Additionally, acute EGFR resistance was modeled using a single exposure to EGFR TKI afatinib or neratinib in vitro over a 48-hour time course. Preliminary multiomic characterization of these data has revealed several targets that can be further investigated for therapeutic exploitation. Further investigation into these targets using orthotopic allografts with CEv3 cells shows that combinatorial therapy with neratinib and abemacilib, a CDK inhibitor, significantly (p < 0.001, n= 20 mice per group) extends survival (56 days) compared to neratinib alone (31.5 days). Future integrated multiomic analysis aims to elucidate the synergistic relationship between neratinib and abemacilib.

INNV-18. FOCUS ON CURRENT AND EMERGING TREATMENT OPTIONS FOR GLIOMA: A COMPREHENSIVE REVIEW

Abstract This comprehensive review delves into the current updates and challenges associated with the management of low-grade gliomas (LGG), the predominant primary tumors in the central nervous system. With a general incidence rate of 5.81 per 100000, gliomas pose a significant global concern, necessitating advancements in treatment techniques to reduce mortality and morbidity. This review places a particular focus on immunotherapies, discussing promising agents such as Zotiraciclib and Lerapolturev. Zotiraciclib, a CDK9 inhibitor, has demonstrated efficacy in glioblastoma treatment in preclinical and clinical studies, showing its potential as a therapeutic breakthrough. Lerapolturev, a viral immunotherapy, induces inflammation in glioblastoma and displays positive outcomes in both adult and pediatric patients. Exploration of immunotherapy extends to Pembrolizumab, Nivolumab, and Entrectinib, revealing the challenges and variabilities in patient responses. Despite promising preclinical data, the monoclonal antibody Depatuxizumab has proven ineffective in glioblastoma treatment, emphasizing the critical need to understand resistance mechanisms. The review also covers the success of radiation therapy in pediatric LGG, with evolving techniques, such as proton therapy, showing potential improvements in patient quality of life. Surgical treatment is discussed in the context of achieving a balance between preserving the patient’s quality of life and attaining gross total resection, with the extent of surgical resection significantly influencing the survival outcomes. In addition to advancements in cancer vaccine development, this review highlights the evolving landscape of LGG treatment, emphasizing a shift toward personalized and targeted therapies. Ongoing research is essential for refining strategies and enhancing outcomes in the management of LGG.

Aurora B inhibitors promote RB hypophosphorylation and senescence independent of p53-dependent CDK2/4 inhibition

Aurora B kinase (AURKB) inhibitors have been trialled in a range of different tumour types but are not approved for any indication. Expression of the human papilloma virus (HPV) oncogenes and loss of retinoblastoma (RB) protein function has been reported to increase sensitivity to AURKB inhibitors but the mechanism of their contribution to sensitivity is poorly understood. Two commonly reported outcomes of AURKB inhibition are polyploidy and senescence, although their relationship is unclear. Here we have investigated the major cellular targets of the HPV E6 and E7, p53 and RB, to determine their contribution to AURKB inhibitor induced polyploidy and senescence. We demonstrate that polyploidy is a universal feature of AURKB inhibitor treatment in all cell types including normal primary cells, but the subsequent outcomes are controlled by RB and p53. We demonstrate that p53 by regulating p21 expression is required for an initial cell cycle arrest by inhibiting both CDK2 and CDK4 activity, but this arrest is only triggered after cells have undergone two failed mitosis and cytokinesis. However, cells can enter senescence in the absence of p53. RB is essential for AURKB inhibitor-induced senescence. AURKB inhibitor induces rapid hypophosphorylation of RB independent of inhibition of CDK2 or CDK4 kinases and p53. This work demonstrates that p53 activation determines the timing of senescence onset, but RB is indispensable for senescence.

Cyclin-Dependent Kinase Inhibitors in the Rare Subtypes of Melanoma Therapy.

Melanoma occurs in various forms and body areas, not only in the cutis, but also in mucous membranes and the uvea. Rarer subtypes of that cancer differ in genomic aberrations, which cause their minor sensibility to regular cutaneous melanoma therapies. Therefore, it is essential to discover new strategies for treating rare forms of melanoma. In recent years, interest in applying CDK inhibitors (CDKIs) in cancer therapy has grown, as they are able to arrest the cell cycle and inhibit cell proliferation. Current studies highlight selective CDK4/6 inhibitors, like palbociclib or abemaciclib, as a very promising therapeutic option, since they were accepted by the FDA for advanced breast cancer treatment. However, cells of every subtype of melanoma do not react to CDKIs the same way, which is partly because of the genetic differences between them. Herein, we discuss the past and current research relevant to targeting various CDKs in mucosal, uveal and acral melanomas. We also briefly describe the issue of amelanotic and desmoplastic types of melanoma and the need to do more research to discover cell cycle dysregulations, which cause the growth of the mentioned forms of cancer.

Combinatorial Therapy of CDK9 Inhibitor with CD19 CAR-T to Reciprocally Overcome Therapy Resistance and Enhance Treatment Efficacies Against Aggressive B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are subgroups of aggressive B-cell lymphoma associated with unacceptably high rates of refractoriness to intensive treatment and significantly shortened survival in this setting. In addition to standard therapies, modern lymphoma care has incorporated targeted agents including Bruton's tyrosine kinase inhibitors and BCL-2 antagonists. Despite achieving initial clinical responses, many patients eventually succumb to disease relapse following treatment with these novel agents. Meanwhile, chimeric-antigen receptor (CAR) T cell therapies have produced unprecedented initial responses in patients with relapsed or refractory DLBCL and MCL, yet at follow-up, only 40% of patients are able to sustain durable remissions. Thus, there is an urgent need to improve the duration of response and delay or prevent CAR-T therapy resistance. To this end, we previously performed mechanistic and translational studies aimed at developing CDK9 as a novel therapeutic target in DLBCL and MCL. This work involved the interrogation of gene expression and functional screening of a large panel of primary patient samples and lymphoma cell lines, ultimately revealing an essential transcription state characterized by increased RNA polymerase II-mediated transcription as the primary driver of therapy resistance evolution and fatal disease progression. Although clinical trials of CDK9 inhibitors (CDK9i) in B-cell lymphomas have shown promise in drug and CAR-T resistant lymphoma patients, mono-targeting of CDK9 has encountered clinical challenges, particularly the development of resistance in initial responders. Thus, understanding CDK9i resistance evolution mechanisms and identifying ways to target the key resistance-associated molecular determinants and subpopulation(s) of cells capable of giving rise to resistant disease holds great potential for advancing lymphoma therapy. Here, we extend our previous work with CDK9i to identify the underlying molecular determinants of CDK9i resistance and, ultimately, to design rational combination therapies to enhance and sustain CDK9i efficacy. In doing so, we are able to demonstrate that: 1) DLBCL and MCL are exquisitely sensitive to CDK9 inhibition (NVP2/AZD4573) regardless of genetic background or resistance status, 2) populations of CDK9i (NVP2/AZD4573) treatment-induced drug tolerant persister (DTP) cells are responsible for driving therapy resistance evolution, 3) CDK9i (NVP2/AZD4573) treatment induces an immunogenic response via activation of proinflammatory and IFN pathways ex vivo and in vivo, and 4) CDK9i and CD19 CAR-T therapies exhibit reciprocally enhanced efficacy in a manner that can overcome therapy resistance in DLBCL and MCL. The outcomes of this study have broad applicability across B-cell malignancies for eradicating both targeted and CAR-T therapy resistance, which we anticipate can be readily translated to the clinic and have immediate impact on DLBCL/MCL patient care.

Overcoming Stress Response-Induced Drug Resistance through Targeting CDK9 in Ph+ Leukemia

Introduction: The toxicity and quality of life burden following polychemotherapy protocols are significant, even among pediatric ALL patients classified as “low risk.” Therefore, it is essential to look for alternative therapies for patients with high-risk features, such as Philadelphia chromosome-positive (Ph+) leukemia, where harsh treatments are usually required. Heat shock protein 90 (HSP90), involved in stress response, is a critical target in leukemia due to its significant upregulation and essential role in stabilizing numerous oncogenic proteins, such as BCR::ABL1 kinase. However clinical approval of HSP90 inhibitors (HSP90i) is hampered due to resistance mechanisms, such as the heat shock response (HSR). Our recent findings identified HSP90α as the principal contributor to resistance in Ph+ leukemia cells. The combination of CDK7i and HSP90i synergistically inhibited the HSR via RNA Polymerase II (RNAPII) in Ph+ leukemia cells resistant to multiple TKIs.1 Methods: The present study investigated the effects of HSP90 and CDK9 inhibitors on Ph+ leukemia cells. Conventional and capillary-based (JESS) Western blotting assessed HSR- and apoptosis-related protein expression following HSP90i and CDK9i treatment. Isogenic cell line models overexpressing anti-apoptotic proteins MCL-1, BCL-2, and BCL-xL were generated. Time-dependent qPCR validated transcriptional changes in HSR- and apoptosis-related genes post-treatment. Matrix synergy drug screening was performed to identify synergistic drug interactions in Ph+ leukemia cell lines and patient-derived xenograft (PDX) leukemia cells. Results: We investigated the pivotal role of CDK9, a crucial mediator in the transcriptional elongation of stress response genes and downstream of CDK7 in regulating RNAPII mediated transcription. Concurrent inhibition of CDK9 and HSP90 effectively diminishes HSP90AA1 expression, a key chaperone in cellular stress responses. The co-targeting strategy disrupted the induction of the HSR by specifically downregulating HSP90α (p = 0.0003) and HSP70 (p < 0.0001) at the protein level, along with downregulating HSP90α/β, HSP70, HSP40, and HSP27 at the transcriptional level. A notable consequence of this dual inhibition is the downregulation of short-lived oncogenic proteins such as c-Myc and Mcl-1 over a time period of 1-24 hours. Additionally, other anti-apoptotic members of the BCL-2 family, including BCL-2 and BCL-xL are also downregulated within 6-24 hours after the treatment. This reduction in anti-apoptotic proteins correlates with an increase in cleaved Poly (ADP-ribose) polymerase (cl-PARP) and apoptotic cells (% sub-G1; pHSP90i:Combi < 0.0001; pCDK9i:Combi = 0.0001). Notably the combination of CDK9 and HSP90 inhibition is equally effective in isogenic leukemia cell line models, overexpressing BCL2 family members (BCL2, BCL-xl, and MCL-1). The synergistic efficacy of CDK9i and HSP90i has been validated across various leukemia cell lines (maximum ZIP score: >15; N=12) and patient-derived leukemia cells (N=3), highlighting its potential therapeutic significance in combating resistance. Summary: Dual targeting of HSP90 and CDK9 achieves HSR abrogation through RNAPII inhibition, leading to disruption of the transcription machinery responsible for activating stress response genes. Further, the combination will be tested in preclinical leukemia models, both alone and in conjunction with standard chemotherapy, with the goal of reducing doses and achieving stable remissions. 1. Vogt, M.; Dienstbier, N. et al. Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells. Cell Death & Disease 2023, 14 (12), 799.

High Remission Rates in Relapsed/Refractory Acute Myeloid Leukemia with QHRD107 (CDK9 Inhibitor), Venetoclax, and Azacitidine Combination Therapy (107VA regimen) : Preliminary Results of a Phase 2a Study

High Remission Rates in Relapsed/Refractory Acute Myeloid Leukemia with QHRD107 (CDK9 Inhibitor), Venetoclax, and Azacitidine Combination Therapy (107VA regimen) : Preliminary Results of a Phase 2a Study

Preliminary Results of a Phase 1, Dose-Escalation Study of PRT2527, a Potent and Highly Selective CDK9 Inhibitor, As Monotherapy and in Combination with Zanubrutinib in Patients with Relapsed/Refractory Lymphoid Malignancies

Preliminary Results of a Phase 1, Dose-Escalation Study of PRT2527, a Potent and Highly Selective CDK9 Inhibitor, As Monotherapy and in Combination with Zanubrutinib in Patients with Relapsed/Refractory Lymphoid Malignancies