Cyclin-dependent Kinase 9 Inhibitor Research Articles

Abstract PO4-18-07: A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in triple negative breast cancer and transcriptionally addicted relapsed or refractory solid tumors

Abstract Background: MYC deregulation is a hallmark of triple negative breast cancer (TNBC) and is associated with aggressive tumors and poor clinical outcomes. Although MYC remains undrugged, targeting of its cofactors has emerged as an attractive strategy to inhibit MYC oncogenic activity. Cyclin-dependent kinase 9 (CDK9) is a critical regulator of oncogenic MYC expression and an important MYC cofactor. KB-0742 is an oral CDK9 inhibitor that demonstrates promising preclinical activity against TNBC. In a real-world cohort, TNBCs have higher MYC expression and higher rates of MYC genomic amplification than other breast cancer subtypes. In primary patient-derived cell lines, KB-0742 treatment results in stronger cytotoxic effects in TNBC as compared to other subtypes. In patient-derived organoids and patient-derived xenografts, CDK9 inhibition by KB-0742 drives antiproliferative and anti-tumor growth effects, including in models that are resistant to standard of care. KB-0742 strongly downregulates MYC protein levels at doses that only partially inhibit CDK9 activity, suggesting a therapeutic window for tumor-specific activity (Saffran, D.C. et al, 2021). KB-0742 is currently being evaluated in a phase 1/2 dose escalation and cohort expansion study in patients with TNBC and other transcriptionally addicted tumors (NCT04718675). Trial design: This phase 1/2 study includes two parts: dose escalation (part 1) and cohort expansion (part 2). Part 2 includes cohort A (solid tumors with high prevalence of MYC overexpression including TNBC, non-small cell lung cancer and ovarian) and cohort B (other transcriptionally addicted tumor types including sarcomas, adenoid cystic carcinoma, nut midline carcinoma and small cell lung cancer). KB-0742 is dosed orally once daily for 3 consecutive days, followed by 4 days, off on a weekly basis in 28-day cycles until unacceptable toxicity or disease progression. Eligibility criteria: Part 1 dose escalation is open to patients with relapsed or refractory solid tumors. Part 2 is defined by tumor indications in cohorts A and B. Eligibly criteria include age > 18 years (≥ 12 years old and with a body weight ≥ 40 kg part 2 for cohort B), acceptable organ function, and ECOG PS < 2. Specific aims: Primary objectives include evaluation of pharmacokinetics (PK), pharmacodynamics (PD), safety, tolerability, preliminary anti-tumor activity, and identifying a maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). PK measurements include Cmax, tmax, AUC0-last, accumulation ratio (Racc) and t1/2. Safety data will be evaluated per the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0. The Modified Continuous Reassessment Method (mCRM) (Goodman et al., Stat Med 1995) will guide dose escalation and MTD. RP2D nomination is informed by PD in peripheral blood mononuclear cells using assays to evaluate phosphorylation of the CDK9 substrate serine 2 on the RNA Polymerase II C-terminal domain (pSER2) and CDK9-responsive gene expression. Radiographic tumor response to KB-0742 in patients is assessed every other cycle starting from cycle two after treatment using RECIST 1.1 criteria. Target accrual: Targeted total enrollment is 170 patients. For additional information, contact clinicaltrials@kronosbio.com Citation Format: Monica Mita, Alain Mita, Miguel Villalona-Calero, Noah Federman, Drew Rasco, David Spigel, Jia Luo, Gregory Cote, Richard Cutler, Pavan Kumar, Crystal MacKenzie, Charles Lin, Jorge DiMartino, Elizabeth Olek, Brian Van Tine. A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in triple negative breast cancer and transcriptionally addicted relapsed or refractory solid tumors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-18-07.

Abstract PO3-04-12: SUMIT-BC: Phase 2 randomized study of fulvestrant with or without the cyclin-dependent kinase 7 inhibitor (CDK7i) samuraciclib in advanced hormone receptor positive (HR+) breast cancer after CDK4/6i

Abstract Background: CDK7 has 3 critical roles in cancer: enhanced transcription of oncogenes and upregulation of anti-apoptotic genes, acceleration of the cell cycle through phosphorylation of other CDKs and driving estrogen receptor (ER) resistance to hormonal therapy. Inhibition of CDK7 is therefore a potential novel anti-cancer therapeutic strategy. In an initial single-arm evaluation samuraciclib (CT7001), a once daily (QD) oral CDK7 inhibitor has demonstrated a favourable safety profile and clinical activity in combination with fulvestrant, a selective estrogen receptor degrader (SERD), in patients with HR+/HER2- advanced breast cancer who have previously been treated with a CDK4/6 inhibitor. There was evidence of enhanced benefit in patients with no detectable TP53 mutation from baseline circulating tumor DNA (ctDNA) analysis and in patients without baseline liver metastases [1]. Samuraciclib is associated with low grade gastrointestinal (GI) adverse events such as nausea, vomiting and diarrhea managed with simple prophylaxis and patient counselling. An instant release capsule formulation was used in the initial clinical evaluation of samuraciclib requiring patients to take multiple capsules designed to rapidly release a large amount of material high in the GI tract. In this study a novel single tablet formulation will be administered which may enhance GI tolerability (2). This Phase 2 open-label randomised study will evaluate the efficacy, safety, pharmacokinetics and Quality of Life (QoL) impact of samuraciclib in combination with fulvestrant in comparison to a fulvestrant monotherapy control arm. Consistent with the principles of the current Project OPTIMUS initiative, 2 dose levels of samuraciclib will be evaluated [3]. All patients will undergo baseline ctDNA evaluation of TP53 mutation status to permit a prospective evaluation of its patient selection biomarker potential. Trial Design: Eligible patients will provide written informed consent, be aged 18 years or older, have histologically or cytologically confirmed ER+/HER2- advanced or metastatic breast cancer not amenable to resection or radiotherapy of curative intent, have received an aromatase inhibitor in combination with a CDK4/6 inhibitor in either the adjuvant or advanced setting, be receiving a luteinizing hormone-releasing hormone agonist if pre/perimenopausal and have RECIST v1.1 evaluable disease. Prior SERD, mammalian target of rapamycin inhibitor (mTORi) or chemotherapy for advanced breast cancer are not permitted. All patients will undergo baseline Guardant360 ctDNA analysis to establish their TP53 mutation status among others. 60 patients will be enrolled and will all receive fulvestrant 500mg IM administered on days 1, 15 and 29 and then monthly thereafter. Patients will be randomized 1:1:1 to fulvestrant alone, fulvestrant and samuraciclib 240mg QD or fulvestrant and samuraciclib 360mg QD. Patients will undergo RECIST v1.1 evaluation at baseline every 8-weeks until week 48, followed by every 12-weeks thereafter. The pharmacokinetics of samuraciclib and fulvestrant will be studied though the first 6 months of the study, with highest intensity during month 1. To evaluate Quality of Life (QoL) The Functional Assessment of Cancer Therapy -Breast questionnaire (FACT-B) will be completed. The primary endpoint is clinical benefit response at 24 weeks. Secondary endpoints are tolerability, progression free survival, overall response rate, duration of response, and the pharmacokinetics of fulvestrant and samuraciclib. QoL and correlations between ctDNA detectable TP53 mutations and efficacy/safety finding in this patient population will be reported. The study opened for recruitment in June 2023.

Abstract PO3-04-13: SUMIT-ELA: Phase 1b/2 combination of cyclin-dependent kinase 7 inhibitor (CDK7i) samuraciclib and selective estrogen receptor degrader (SERD) elacestrant in advanced hormone receptor positive (HR+) breast cancer after CDK4/6i

Abstract Background: CDK7 has 3 critical roles in cancer: enhanced transcription of oncogenes and upregulation of anti-apoptotic genes, acceleration of the cell cycle through phosphorylation of other CDKs and driving estrogen receptor (ER) resistance to hormonal therapy. Inhibition of CDK7 is therefore a potential novel anti-cancer therapeutic strategy. Samuraciclib (CT7001), a once daily oral CDK7 inhibitor has demonstrated a favorable safety profile and clinical activity in combination with fulvestrant (SERD) in patients with HR+/HER2- advanced breast cancer who have previously been treated with a CDK4/6 inhibitor [1]. Extended benefit was observed in patients with no TP53-mutation per circulating tumor DNA (ctDNA) analysis detectable at baseline. Limitations in the pharmacokinetic (PK) properties and route of administration (intramuscular only) of fulvestrant mean that oral alternatives are desirable. Recently, the EMERALD phase 3 study of the oral SERD elacestrant demonstrated a significant PFS benefit over standard of care endocrine treatment in the ER+/HER2- advanced or metastatic breast cancer population previously treated with a CDK4/6 inhibitor. [2]. Non-clinical data indicate that the underlying biology driving samuraciclib combination with endocrine therapy translates from fulvestrant to elacestrant, clinical evaluation is warranted [3]. This open-label Phase 1b dose escalation and Phase 2 dose expansion study will evaluate the safety, efficacy and pharmacokinetics of samuraciclib in combination with elacestrant. ctDNA analysis is informative for both elacestrant and samuraciclib, via ESR1-mutation status and TP53-mutation status respectively, and is an integral part of the study design. Trial Design: Eligible patients will provide written informed consent, be aged 18 years or older, have histologically or cytologically confirmed ER+/HER2- advanced or metastatic breast cancer not amenable to resection or radiotherapy of curative intent, have received an aromatase inhibitor in combination with a CDK4/6 inhibitor in either the adjuvant or advanced setting, be receiving a luteinizing hormone-releasing hormone agonist if pre/perimenopausal and have RECIST v1.1 evaluable disease. Prior SERD, mammalian target of rapamycin inhibitor (mTORi) or chemotherapy for advanced breast cancer are not permitted. The study will enrol ~48 patients. All patients will undergo baseline Guardant360 ctDNA analysis to establish their ESR-1 and TP53-mutation status. Patients will undergo RECIST v1.1 evaluation at baseline every 8-weeks until week 48, followed by every 12-weeks thereafter. Initially both samuraciclib and elacestrant will be administered once daily in a dose escalation/de-escalation approach under supervision of the study Safety Review Committee. The primary endpoint in Phase 1b is the identification of tolerable combination dose levels for both samuraciclib and elacestrant and in Phase 2 expansion to quantify the progression free survival benefit of the combination. Secondary endpoints are tolerability, clinical benefit response at 24 weeks, overall response rate, duration of response, best percent change in tumor size, pharmacokinetics and correlations between ctDNA detectable ESR1 and TP53 mutations and efficacy/safety finding in this patient population. The study opened for recruitment in June 2023.

Design, synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel potent CDK7 inhibitors

The targeting of cyclin-dependent kinase 7 (CDK7) has become a highly desirable therapeutic approach in the field of oncology due to its dual role in regulating essential biological processes, encompassing cell cycle progression and transcriptional control. We have previously identified a highly selective thieno[3,2-d]pyrimidine-based CDK7 inhibitor with demonstrated efficacy and safety in animal model. In this study, we sought to optimize the thieno[3,2-d]pyrimidine core to discover a novel series of CDK7 inhibitors with improved potency and pharmacokinetic (PK) properties. Through extensive structure–activity relationship (SAR) studies, compound 20 has emerged as the lead candidate due to its potent inhibitory activity against CDK7 and remarkable efficacy on MDA-MB-453 cells, a representative triple negative breast cancer (TNBC) cell line. Furthermore, 20 has demonstrated favorable oral bioavailability and exhibited highly desirable pharmacokinetic (PK) properties, making it a promising lead candidate for further structural optimization.

Inhibition of CDK7 mitigates doxorubicin cardiotoxicity and enhances anticancer efficacy.

The anthracycline family of anticancer agents such as doxorubicin (DOX) can induce apoptotic death of cardiomyocytes and cause cardiotoxicity. We previously reported that DOX-induced apoptosis is accompanied by cardiomyocyte cell cycle-reentry. Cell cycle progression requires cyclin-dependent kinase 7 (CDK7)-mediated activation of downstream cell cycle CDKs. This study aims to determine whether CDK7 can be targeted for cardioprotection during anthracycline chemotherapy. DOX exposure induced CDK7 activation in mouse heart and isolated cardiomyocytes. Cardiac-specific ablation of Cdk7 attenuated DOX-induced cardiac dysfunction and fibrosis. Treatment with the covalent CDK7 inhibitor THZ1 also protected against DOX-induced cardiomyopathy and apoptosis. DOX treatment induced activation of the proapoptotic CDK2-FOXO1-Bim axis in a CDK7-dependent manner. In response to DOX, endogenous CDK7 directly bound and phosphorylated CDK2 at Thr160 in cardiomyocytes, leading to full CDK2 kinase activation. Importantly, inhibition of CDK7 further suppressed tumor growth when used in combination with DOX in an immunocompetent mouse model of breast cancer. Activation of CDK7 is necessary for DOX-induced cardiomyocyte apoptosis and cardiomyopathy. Our findings uncover a novel proapoptotic role for CDK7 in cardiomyocytes. Moreover, this study suggests that inhibition of CDK7 attenuates DOX-induced cardiotoxicity, but augments the anticancer efficacy of DOX. Therefore, combined administration of CDK7 inhibitor and DOX may exhibit diminished cardiotoxicity but superior anticancer activity.

Abstract CT158: A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in relapsed, refractory and transcriptionally addicted solid tumors

Abstract Background MYC transcriptional deregulation is a hallmark of cancer and is associated with poor clinical outcomes. MYC deregulated solid tumors including non-small cell (NSCLC), small cell lung cancer (SCLC), triple negative breast cancer (TNBC), and ovarian cancer are known for their aggressiveness and frequent relapse. Analysis of a real-world solid tumor cohort including NSCLC (n= 20,470), SCLC (n=1,517), TNBC (n=2,576) and ovarian cancer (n=1,667) demonstrated MYC family overexpression and/or genomic amplification in 47% to 87% of patients. Although MYC remains difficult to drug, targeting of its cofactors has emerged as an attractive strategy to inhibit MYC oncogenic activity. Cyclin-dependent kinase 9 (CDK9), a MYC cofactor, is a critical regulator of oncogenic MYC expression and activity. KB-0742 is a potent, selective, and orally bioavailable inhibitor of CDK9 with a long plasma half-life. KB-0742 is being evaluated in an ongoing Phase 1/2 study in advanced solid tumors (NCT04718675). Methods The Phase 1/2 study is being conducted in two parts: dose escalation and dose expansion. Part 1 dose escalation comprises patients with relapsed or refractory solid tumors. Part 2 dose expansion comprises patients with solid tumors that have a high prevalence of MYC overexpression including ovarian cancer, NSCLC, TNBC and SCLC. KB-0742 is dosed orally once daily for 3 consecutive days followed by 4 days off on a weekly basis in 28-day cycles until unacceptable toxicity or disease progression. Eligibility criteria include age > 18 years acceptable organ function and ECOG PS < 2. Up to 170 patients are planned to be enrolled in the study. Primary objectives include evaluation of pharmacokinetics (PK), pharmacodynamics (PD), safety, tolerability, and preliminary anti-tumor activity with the goal of identifying a maximum tolerated dose (MTD). Plasma PK measurements include Cmax, tmax, AUC0-last, accumulation ratio (Racc) and t1/2. Safety data will be evaluated per the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0. RP2D is informed by PD in peripheral blood mononuclear cells using assays to evaluate phosphorylation of the CDK9 substrate serine 2 on the RNA Polymerase II C-terminal domain (pSER2) and CDK9-responsive gene expression. Radiographic tumor response is assessed using RECIST 1.1 criteria. Exploratory objectives include assessment of KB-0742 PD in tumor tissue and profiling of treatment-related genomic, transcriptomic, and proteomic changes. The study is continuing as planned; the next data analysis will occur in 2024. Citation Format: Miguel Villalona Calero, Mark Agulnik, Monica Mita, Alain Mita, Noah Federman, Drew Rasco, David Spigel, Jia Luo, Glenn Hanna, Gregory Cote, Mohamad A. Salkeni, Rashmi Chugh, Natraj R. Ammakkanavar, Satish A. Shah, Amol Rao, Kamalesh Kumar Sankhala, Richard E. Cutler, Tressa Hood, Luis Carvajal, Crystal MacKenzie, Charles Lin, Jorge DiMartino, Elizabeth A. Olek, Brian Van Tine. A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in relapsed, refractory and transcriptionally addicted solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT158.

Selective CDK7 Inhibition Suppresses Cell Cycle Progression and MYC Signaling While Enhancing Apoptosis in Therapy-resistant Estrogen Receptor-positive Breast Cancer.

Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms. We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models. We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models. Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments.

Abstract 1218: CDK9 inhibitors modulate the transcriptional landscape of colorectal cancer to suppress MAPK signaling and synergizes with BRAF inhibitors to treat BRAF-mutant colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US, and it remains a significant public health burden. Metastatic CRC (mCRC) is usually treated with combination chemotherapy and targeted therapy regimens, but eventually becomes chemotherapy refractory. Thus, novel and more effective treatments for mCRC are urgently needed. Cyclin-dependent kinase 9 (CDK9) is a key activator of RNA Pol II transcription and promotes the expression of many cancer driver genes, making CDK9 a promising target for cancer therapy. Methods: Human CRC cell lines, patient-derived organoids (PDOs), cell line xenografts, and patient-derived xenografts were used to investigate the efficacy and mechanisms of action of the CDK9 inhibitors AZD4573, enitociclib, and NVP-2, as well as the BRAF inhibitors encorafenib and dabrafenib. In vitro viability was measured by chemical cell proliferation assays and in vivo tumor sizes were measured by calipers. In vitro mechanisms of action were assessed by RNA sequencing, real time polymerase chain reaction, and Western blotting. In vivo mechanisms of action were assessed by immunohistochemistry. Results: CDK9 inhibitors potently inhibited CRC cells and PDOs. In contrast to hematologic malignancies, we did not observe consistent suppression of the oncogenes c-MYC and MCL-1 by CDK9 inhibitor treatment. Instead, CDK9 inhibitors suppressed several other key cancer pathways in CRC, such as MAPK, mTOR, and PI3K signaling pathways. Importantly, multiple targets within the MAPK pathway were strongly suppressed, including EGFR, KRAS, and BRAF. We hypothesized that combination treatment with CKD9 inhibitors and MAPK pathway inhibitors can synergistically treat CRC. As proof-of-concept, we investigated the combination of CDK9 and BRAF inhibitors in models of BRAF-mutant CRC, a particularly aggressive type of CRC. We found this combination to synergistically suppress CRC growth in vitro and in vivo. Compared to single agents, combination treatment led to significantly stronger induction of apoptosis and suppression of MAPK pathway signaling. Our results suggest that concurrent treatment with CDK9 inhibitors plus established MAPK pathway inhibitors, such as BRAF inhibitors, can significantly improve upon single agent treatment. Conclusions: We have found CDK9 inhibitors to potently suppress CRC growth and survival through a unique mechanism of action, by vertical suppression of the MAPK signaling pathway. We demonstrate that CDK9 inhibitors can synergize with BRAF inhibitors in the treatment of BRAF-mutant CRC models. Thus, CDK9 inhibitors are a promising class of drugs warranting further investigation, including early-phase clinical trials, in mCRC. Citation Format: Chaoyuan Kuang, Ning Wei, Mahshid Mohammadi, Muzaffer A. Bhat, Terence Li, Priyanka Patil, Othon Wiltz, Renee Huang, Kohtaro Ooka, Melanie Quintal, Edward Chu. CDK9 inhibitors modulate the transcriptional landscape of colorectal cancer to suppress MAPK signaling and synergizes with BRAF inhibitors to treat BRAF-mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1218.

Abstract 528: CDK9 inhibition activates innate immune response in prostate cancer cells

Abstract Hyper-activation of transcription is frequent in cancer, which often leads to increased sensitivity to compounds targeting the transcriptional kinases, in particular cyclin-dependent kinase 9 (CDK9). However, mechanistic details as to why CDK9 inhibition selectively kills cancer cells remain largely unknown. Here, we report that CDK9 inhibition activates innate immune response through viral mimicry. In MYC over-expressing prostate cancer cells, CDK9 inhibition leads to excessive accumulation of mis-spliced RNA. Double-stranded RNA (dsRNA)-activated kinase can recognize these mis-spliced RNAs, and we show that this kinase is required for the CDK9 inhibitor-induced anti-proliferative effects. Using time-resolved transcriptional profiling (SLAM-seq), targeted proteomics and ChIP-seq, we show that, similar to viral infection, CDK9 inhibition significantly suppresses transcription of most genes but allows selective transcription and translation of certain genes. In particular, CDK9 inhibition activates NFκB-driven cytokine-signaling at the transcriptional- and secretome-levels. Transcriptional signature induced by CDK9 inhibition identifies prostate cancers with high level of genome instability, and we propose that it is possible to induce similar effects using CDK9 inhibitors. In summary, here we show that inhibition of CDK9 activates innate immune response through viral mimicry. In the future, it is important to establish if CDK9 inhibitors can potentiate the effects of immunotherapy against the late-stage prostate cancer, a currently lethal disease. Citation Format: Shivani Yalala, Aishwarya Gondane, Ninu Poulose, Jing Liang, Ian G. Mills, Harri M. Itkonen. CDK9 inhibition activates innate immune response in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 528.

Abstract 5707: Preclinical evaluation of KRLS-017, a potent, highly selective and reversible CDK7 inhibitor with broad antitumor effect in preclinical models, in preparation for a Phase 1 clinical trial in advanced solid tumor malignancies

Abstract KRLS-017 is a potent, selective, and reversible inhibitor of Cyclin Dependent Kinase 7 (CDK7). Its molecular formula is C24H35N5O2Si and its molecular weight is 453.65 Daltons. KRLS-017 is being developed for the treatment of patients with advanced solid tumor malignancies. To differentiate KRLS-017 from other CDK7 inhibitors, we created a reversible inhibitor to allow for treatment schedule optimization and maximization of therapeutic index in clinic. High selectivity of KRLS-017 for CDK7 was confirmed using a panel of 313 recombinant human kinases. The IC50 of KRLS-017 against recombinant human CDK7 was determined to be 16 nM, and no other kinases in the panel showed IC50 values less than 300 nM. Antiproliferative activity of KRLS-017 was investigated in vitro using a broad panel of human tumor cell lines. KRLS-017 showed potent antiproliferative effect across a wide variety of both solid and hematologic tumor types, producing GI50 values <200 nM in 258/302 human tumorcell lines tested. Antitumor activity of KRLS-017 was investigated in vivo using multiple mouse xenograft tumormodels at dose levels ranging from 25 mg/kg to 200 mg/kg using both continuous (daily) andintermittent dosing schedules. Statistically significant tumor growth inhibition was seen at doselevels of 50 mg/kg and higher, with tumor regression observed at 100 mg/kg and 200 mg/kg. Better in vivo tolerability at efficacious doses was observed using intermittent dosing schedules in mouse xenograft models, which led to adoption of an intermittent schedule in the proposed first-in-human phase 1 trial. KRLS-017 is orally bioavailable in mice, rats, and dogs and plasma drug exposure increased in a proportional manner with dose. Comparing first dose and 28th dose in the dog, minimal plasma accumulation was observed, suggesting once daily dosing in humans would be initially tested. Repeat dose 28 day GLP toxicology studies in rat and dog showed adverse effects in highly proliferative bone marrow and gastrointestinal tissues which is consistent with the known mechanism of action for CDK7 and is considered to be monitorable and manageable in the setting of oncology. The clinical development plan for KRLS-017 includes an initial Phase 1 dose escalation study (KRLS-017-101) in patients with refractory solid tumor malignancies to assess pharmacokinetics/pharmacodynamics and to determine an optimal dose level and treatment schedule to test in expansion cohorts. Given the potential of CDK7 inhibitors in the treatment of hormone receptor positive breast cancer, combination work with other agents (such as selective estrogen receptor degraders) is on-going. Citation Format: Alison L. Hannah, Steve Ritland, Thomas Steele, Steven Smith, Ashwin Ram, BT Slingsby, Kapil Dhingra, James Chrisman, Rajesh Dua, Yasunori Tokunaga, Shigeyuki Kono, Yasuhiro Aga. Preclinical evaluation of KRLS-017, a potent, highly selective and reversible CDK7 inhibitor with broad antitumor effect in preclinical models, in preparation for a Phase 1 clinical trial in advanced solid tumor malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5707.

Abstract 3197: Addressing drug metabolism and pharmacokinetics (DMPK) challenges of small molecule-drug conjugates (SMDCs)

Abstract Background: SMDCs are gaining momentum with potential advantages over antibody-drug conjugates (ADCs), eg, better tumor penetration and lack of immunogenicity. We have developed a novel platform technology with tailored solutions for ADCs and SMDCs with intracellular and extracellular cleavage options. Key features of the SMDC technology comprise of a small molecule ligand such as an αvβ3 integrin binder showing efficient tumor homing and a linker extracellularly cleaved by neutrophil elastase (NE) in the tumor microenvironment allowing for seamless release of different payload classes.1 Our lead SMDC VIP2362 is currently in a Phase 1 clinical trial for the treatment of advanced solid tumors (NCT05712889). SMDCs often have a shorter half-life compared with ADCs, which may result in more frequent dosing schedules. Here we show significant reduction of clearance and drastic prolongation of half-life of SMDCs achieved by adding a second binding moiety. Methods: Payloads such as kinesin spindle protein inhibitors (KSPi), cyclin-dependent kinase 9 inhibitors (CDK9i), and monomethyl auristatin E (MMAE) have been linked via elastase-cleavable linkers to one or, via a branched linker, to two αvβ3 integrin binding moieties. Cytotoxicity of SMDCs was measured in cell lines with or without NE. Monovalent and bivalent SMDCs were investigated in DMPK studies in rats while monitoring the conjugate and the released payload. Results: Payloads were coupled to the NE-cleavable peptides via different linkages. MMAE was conjugated via sterically hindered ester bonds, CDK9i was conjugated via sulfoximide bonds, and KSPi payloads were coupled via amide bonds. Each of these SMDCs was highly stable in plasma and buffer. Without NE, in vitro cytotoxicity of SMDCs in each cell line was poor with IC50 in the micromolar range. In contrast, in the presence of NE, potency increased, and cytotoxicity (IC50) was in the low nanomolar to picomolar range depending on the cell line and payload. In each case, the SMDCs reached similar potency as the free payload indicating efficient cleavage of the SMDC by NE. In DMPK studies of SMDCs in rat, none or trace levels of free payload (<0.1% as compared to the respective conjugates) was measured. With each of the SMDCs and independent of the conjugated payloads, a remarkable impact on exposure was observed when attaching a second binding ligand. The SMDCs with two ligands showed a 37- to 81-fold reduced plasma clearance as compared with the respective monovalent SMDCs, which parallels a 12- to 57-fold increase in half-life (eg, 2.1 h and 28 h for mono and bivalent MMAE SMDCs respectively). Initial in vivo studies in a cell line-derived xenograft model showed efficacy of bivalent SMDCs with once weekly dosing. Conclusion: Our technology platform comprises NE-cleavable SMDCs with promising options to tune the DMPK profile for a longer half-life. 1. Lerchen et al, AACR (2023). 2. Lerchen et al, Cancers (2023). Citation Format: Anne-Sophie Rebstock, Hans-Georg Lerchen, Harvey Wong, Beatrix Stelte-Ludwig, Mareike Wiedmann, Amy J. Johnson, Raquel Izumi, Ahmed Hamdy. Addressing drug metabolism and pharmacokinetics (DMPK) challenges of small molecule-drug conjugates (SMDCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3197.

Abstract 5957: Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer

Abstract Introduction Transcription regulators, such as cyclin-dependent kinase 9 (CDK9), have been implicated in the super-enhancer driven transcriptional control of oncogenes in multiple cancers including pancreatic cancer. CDK9 has been shown to be employed by cancer cells for the constant production of short-lived oncoproteins such as c-Myc to maintain their survival. Due to its critical role in regulating transcription in cancer cells, CDK9 has emerged as a potential therapeutic target. Here, we report the antitumor activity of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, in preclinical models for pancreatic cancer. Experimental Procedures Fragment-based MolecuLern driven AI workflow methods were used to design a series of ATP-competitive CDK9 specific kinase inhibitors. BLX-3030 was selected as one of the most potent leads with an IC50 of 1.1 nM in a cell-free CDK9 kinase assay. A Sulforhodamine B (SRB) assay was used to evaluate the cell growth inhibitory activity of BLX-3030 in a panel of 10 pancreatic ductal adenocarcinoma (PDAC) and 2 pancreatic neuroendocrine tumor (pNET) cell lines. Western blotting was used to determine the effects of BLX-3030 treatment on c-Myc expression in cancer cells. The combination effects between BLX-3030 and standard of care regimen, gemcitabine (GEM) and nab-paclitaxel (NP), was assessed using the SRB assay. Finally, the in vivo antitumor activity of BLX-3030 alone or in combination with GEM and NP was evaluated in a mouse cell line xenograft model for pancreatic cancer. Results BLX-3030 exhibited potent cell growth inhibitory activity in the panel of pancreatic cancer cell lines with IC50 values ranging from 133.5 nM to 508.1 nM. The potency of BLX-3030 in the cell lines appears to correlate with the expression level of c-Myc in the cell lines. BLX-3030 showed similar activities in PDAC and pNET cell lines with mean IC50 values of 244.5 nM and 194.6 nM, respectively. However, treatment with BLX-3030 for 48 hours did not seem to significantly reduce c-Myc expression in PSN-1 PDAC cells. When combined with GEM and NP, BLX-3030 demonstrated mostly additive effects in PSN-1 cells. Preliminary data from the in vivo study show that BLX-3030 improves the antitumor activity of GEM and NP. Conclusion In summary, BLX-3030 potently inhibited the growth of both PDAC and pNET cells in vitro. The growth inhibitory activity of BLX-3030 in PDAC cell lines correlates with their c-Myc expression levels. BLX-3030 showed an additive effect when combined with standard of care drugs (GEM and NP) in PDAC cell lines. Overall, the novel CDK9 inhibitor, BLX-3030, demonstrated potent activity in pancreatic cancer cell line models and presents a promising preclinical lead for pancreatic cancer. (This work was supported in part by Biolexis Therapeutics and the Seena Magowitz Foundation). Citation Format: Yesenia Barrera-Millan, Zhaoliang Li, Hariprasad Vankayalapati, David J. Bearss, Daniel D. Von Hoff, Haiyong Han. Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5957.

Abstract 7550: KB-0742, an oral highly selective CDK9 inhibitor, demonstrates preclinical activity in transcription factor fusion driven adenoid cystic carcinoma patient-derived models

Abstract Adenoid Cystic Carcinoma (ACC) is an aggressive rare type of cancer of the secretory glands with no approved targeted therapy and high unmet clinical need. Deregulated transcription driven by MYB-NFIB or MYBL1-NFIB transcription factor (TF) fusions are a hallmark of ACC pathogenesis. More aggressive disease and resistance to therapy has been associated with co-mutation in NOTCH. Although direct targeting of oncogenic TF fusions has remained challenging, targeting of their activity via transcriptional cofactors has emerged as an attractive and clinically actionable strategy. Cyclin-dependent kinase 9 (CDK9) is a key potentiator of TF activity via its ability to act both as an upstream regulator of TF expression and a downstream cofactor. KB-0742 is a potent, selective, and orally bioavailable inhibitor of CDK9 with a long plasma half-life. KB-0742 is being studied in an ongoing Phase 1/2 study (NCT04718675) in advanced solid tumors including ACC. KB-0742 has demonstrated on-mechanism, single agent anti-tumor activity and a manageable safety profile in heavily pre-treated patients (0-11 prior lines of therapy) with transcriptionally addicted solid tumors. Here we present the preclinical rationale for targeting of oncogenic fusion TF activity in ACC. XenoSTART Patient-Derived Xenograft (XPDX) models were established from viable human tumor tissue for in vivo and ex vivo testing. Cell viability was assessed by CellTiter GLO reagent and phase imaging. Changes in MYB-fusion transcripts were assessed by RNA-Seq. Tumor Fragments (~70 mg) from ACCx5M1, ACCx6, ACCx9, ACCx11 models were implanted into athymic nude mice. Animals were treated with either vehicle (normal saline) or 60 mg/kg KB-0742 on a Q3D/week until tumors reached 2500 mm3 or up to 60 days post start of treatment. In a retrospective analysis using real world data, ACC has a low incidence of genomic instability and high rates of MYB fusions detectable by RNA-seq, making immunotherapy challenging but creating an opportunity for targeting MYB transcription. In primary MYB-fusion positive and NOTCH co-mutated patient-derived spheroid models, KB-0742 treatment induced strong antiproliferative activity and cytotoxicity. Importantly, KB-0742 demonstrated stronger cytotoxic effects when compared to historical control agents. In XPDXs, CDK9 inhibition with KB-0742 resulted in antiproliferative activity and stronger tumor growth inhibition in MYB-fusion positive and NOTCH co-mutated tumor models compared to MYB-fusion positive only models.These data demonstrate KB-0742 is effective in preclinical models of ACC suggesting KB-0742 may be a promising therapeutic option for ACC patients. KB-0742 is currently being evaluated in a phase 1/2 dose-escalation and cohort expansion trial in patients with ACC and other transcriptionally addicted tumors (NCT04718675). Citation Format: Michael R. McKeown, Luis A. Carvajal, Tressa R. Hood, Nicole S. Burr, Jeffrey Kaufman, Adam Boynton, Kameron Mori, Tessa DesRochers, Michael Wick, Charles Y. Lin, Jorge F. DiMartino. KB-0742, an oral highly selective CDK9 inhibitor, demonstrates preclinical activity in transcription factor fusion driven adenoid cystic carcinoma patient-derived models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7550.

Abstract 5716: CDK7 as a drug target in advanced stage uveal melanoma

Abstract Uveal melanoma (UM) metastasizes predominantly to the liver, and most metastatic UM are BAP1-deficient. Targeted therapies, such as MEK inhibitors, have failed clinically. There are few FDA-approved therapies for metastatic UM, but these therapies are limited to only a subset of patients and have modest survival benefits. Hence, there is a need to identify novel and effective treatment strategies. Due to the low mutational burden in UM, targeting transcriptional may be an effective strategy. Here, we identified Cyclin Dependent Kinase 7 (CDK7), a key regulator of transcription, as an essential and druggable target in UM. CDK7 directly phosphorylates RNA polymerase II and is expressed in all UMs. siRNA-mediated knockdown of CDK7 decreased the growth of three BAP1-deficient UM cell lines. We tested a clinically relevant, highly specific CDK7 inhibitor, SY-5609, developed by Syros Pharmaceuticals and similarly showed that the cell lines were sensitive to the growth inhibitory effects of SY-5609. Furthermore, we observed synergistic effects on cell growth inhibition when combined with a MEK inhibitor (Trametinib). We performed RNA sequencing and identified several Hallmark Genesets to be positively enriched, including interferon-gamma response, TGF-beta signaling, and NFκB signaling after treatment with SY-5609. These results are validated using a multi-omic approach, utilizing Reverse Phase Protein Array (RPPA) data to validate transcriptome to proteome expression of these enriched pathways. Overall, our studies indicate that targeting CDK7 in uveal melanoma is a potential treatment strategy for BAP1-deficient UM and enhances MEK inhibitor efficacy. Citation Format: Francis J. Waltrich. CDK7 as a drug target in advanced stage uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5716.

Abstract 5966: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies

Abstract CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer cell growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. Specifically, we show that brief treatment with PRT2527 (6h) potently induces apoptosis and inhibits proliferation in DLBCL, MCL and CLL cell lines. We also show that the addition of BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) potentiates the apoptosis response induced by PRT2527 in vitro and leads to more robust and durable responses including complete tumor regressions in DLBCL CDX and PDX models in vivo. In all studies, the combination of PRT2527 with BTKi or BCL2i is well tolerated. Mechanistically, we show that BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency in TMD-8 cells. Concurrent depletion of MCL1 and BFL1 with PRT2527 leads to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In similar fashion, Venetoclax-driven BCL2 inhibition also potentiates the anti-tumor activity of PRT2527. Consistent with these findings in vitro, we observe reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrate potent induction of apoptosis in peripheral blood mononuclear cells isolated from patients with both treatment-naïve and relapse/refractory CLL following brief treatment with PRT2527 (6h). Co-treatment with BTKi or BCL2i further enhances the apoptotic effect of PRT2527, similarly suggesting combinatorial benefit. Additionally, we characterize the activity of PRT2527 in models of BTKi relapsed/refractory MCL where increased CDK9 dependency has previously been described. In an Ibrutinib-resistant Mino CDX model, we observe increased tumor growth inhibition following treatment with PRT2527 compared to tumors derived from parental Mino cells, suggesting PRT2527 is efficacious in BTKi-resistant MCL. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory lymphoid malignancies. Citation Format: Norman Fultang, Ashley M. Schwab, Sophia McAneny-Droz, Diane Heiser, Peggy Scherle, Neha Bhagwat. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5966.

Suppression of cyclin-dependent kinase 1 synergizes with checkpoint kinase 2 inhibitor to promote death of non-small cell lung cancer cells with radiotherapeutic resistance

ObjectiveRadiotherapy resistance poses a prevalent clinical challenge in non-small cell lung cancer (NSCLC), necessitating the development of ongoing treatment protocols following the emergence of radiotherapy resistance, as well as the identification of radiotherapy sensitization drugs. The objective of this study was to exclusively investigate therapeutic targets associated with radiotherapy resistance in NSCLC, thereby expanding the range of treatment alternatives available to patients grappling with radiotherapy resistance. MethodsVarious databases were employed to screen for therapeutic targets associated with radiotherapeutic resistance. The impact of administering inhibitors for cyclin-dependent kinase 1 (CDK1) and checkpoint kinase 2 (CHEK2) on cell proliferation and apoptosis in radioresistant NSCLC cell lines was assessed. ResultsThe potential of CDK1 and CHEK2 kinase as a novel biomarker and therapeutic target in radioresistant NSCLC cells is evident. The up-regulation of CDK1 and CHEK2 expression in NSCLC tissues, as well as their activation in NSCLC cells exposed to X-irradiation, leads to radiotherapeutic resistance. Additionally, the heightened expression of CDK1 and CHEK2 may contribute to an increased score of cancer stem cells (CSCs) in NSCLC patients. The suppression of CDK1 in combination with a CHEK2 inhibitor synergistically promotes the death of NSCLC cells with radiotherapeutic resistance. Notably, the sensitivity of radioresistant NSCLC cell lines to the CHEK2 inhibitor (PV-1019) is enhanced through the pharmacological or genetic inhibition of CDK1. ConclusionsThe current study provides new perspectives on the synergistic effects of CDK1 and CHEK2 inhibitors in inducing apoptosis in NSCLC cells that have acquired resistance to radiotherapy.

Identification of a Novel Selective CDK9 Inhibitor for the Treatment of CRC: Design, Synthesis, and Biological Activity Evaluation.

Cyclin-dependent kinase 9 (CDK9) is a member of the transcription CDK subfamily. In this work, we preliminarily demonstrated the feasibility of CDK9 as a potent target of treatment for colorectal cancer, and a series of novel CDK9 inhibitors were rationally designed and synthesized based on the structure of AZD5438 (a pan CDKs inhibitor reported by AstraZeneca). A novel selective CDK9 inhibitor named CLZX-205, which possessed significant CDK9 inhibitory activity (IC50 = 2.9 nM) with acceptable pharmacokinetic properties and antitumor efficacy in vitro and in vivo, was developed. Research on the mechanism indicated that CLZX-205 could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression, and these results were validated at the cellular and tumor tissue levels. Currently, CLZX-205 is undergoing further research as a promising candidate for CRC treatment.

Abstract B112: A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in relapsed, refractory ovarian cancer and transcriptionally addicted relapsed or refractory solid tumors

Abstract Ovarian cancer is responsible for more female cancer deaths than any other female reproductive malignancy. MYC deregulation is a hallmark of ovarian cancer and is associated with poor clinical outcome. In a real-world cohort (n=1,667), the MYC family of genes is overexpressed and/or genomically amplified in up to 87% of ovarian cancer. Although MYC remains undrugged, targeting of its cofactors has emerged as an attractive strategy to inhibit MYC oncogenic activity. Cyclin-dependent kinase 9 (CDK9) is a critical regulator of oncogenic MYC expression and activity. KB-0742 is an orally bioavailable, highly selective, long half-life (~24 hours) CDK9 inhibitor that demonstrates promising preclinical activity against ovarian cancer and is currently being evaluated in a phase 1/2 dose escalation and cohort expansion study in patients with ovarian cancer and other transcriptionally addicted tumors (NCT04718675). The phase 1/2 study is being conducted in two parts: dose escalation (part 1) and cohort expansion (part 2). Part 2 is comprised of cohort A (solid tumors with high prevalence of MYC overexpression including ovarian cancer, non-small cell lung cancer, and triple negative breast cancer) and cohort B (other transcriptionally addicted tumor types including sarcomas, adenoid cystic carcinoma, nut midline carcinoma and small cell lung cancer). KB-0742 is dosed orally once daily for 3 consecutive days, followed by 4 days off, on a weekly basis in 28-day cycles until unacceptable toxicity or disease progression. It is estimated that up to 170 patients will be enrolled in the study. Part 1 dose escalation is open to patients with relapsed or refractory solid tumors or non-Hodgkin’s lymphoma. Part 2 is defined by tumor indications as described in cohorts A and B. Eligibility criteria include age > 18 years (≥ 12 years old and with a body weight ≥ 40 kg in part 2, cohort B), acceptable organ function, and ECOG PS < 2. Primary objectives include evaluation of pharmacokinetics (PK), pharmacodynamics (PD), safety, tolerability, and preliminary anti-tumor activity, with the goal of identifying a maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Plasma PK measurements include Cmax, tmax, AUC0-last, accumulation ratio (Racc) and t1/2. Safety data will be evaluated per the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0. Dose escalation and MTD identification is guided by a Modified Continuous Reassessment Method (mCRM) (Goodman et al., Stat Med 1995). RP2D is informed by PD in peripheral blood mononuclear cells using assays to evaluate phosphorylation of the CDK9 substrate serine 2 on the RNA Polymerase II C-terminal domain (pSER2) and CDK9-responsive gene expression. Radiographic tumor response is assessed using RECIST 1.1 criteria. Exploratory objectives include assessment of KB-0742 PD in tumor tissue and profiling of treatment-related genomic, transcriptomic, and proteomic changes. Enrollment continues in the United States with sites in Spain, France, and UK expected to open. Citation Format: Miguel Villalona-Calero, Monica Mita, Alain Mita, Noah Federman, Drew Rasco, David Spigel, Jia Luo, Gregory M. Cote, Richard E. Cutler, Pavan Kumar, Crystal J. MacKenzie, Charles Lin, Jorge F. DiMartino, Elizabeth A. Olek, Brian Van Tine. A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in relapsed, refractory ovarian cancer and transcriptionally addicted relapsed or refractory solid tumors [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B112.

Neutrophil elastase as a versatile cleavage enzyme for activation of αvβ3 integrin-targeted small molecule drug conjugates with different payload classes in the tumor microenvironment.

Introduction: The development of bioconjugates for the targeted delivery of anticancer agents is gaining momentum after recent success of antibody drug conjugates (ADCs) in the clinic. Smaller format conjugates may have several advantages including better tumor penetration; however, cellular uptake and trafficking may be substantially different from ADCs. To fully leverage the potential of small molecule drug conjugates (SMDCs) with potent binding molecules mediating tumor homing, novel linker chemistries susceptible for efficient extracellular activation and payload release in the tumor microenvironment (TME) need to be explored. Methods: We designed a novel class of SMDCs, which target αvβ3 integrins for tumor homing and are cleaved by neutrophil elastase (NE), a serine protease active in the TME. A peptidomimetic αvβ3 ligand was attached via optimized linkers composed of substrate peptide sequences of NE connected to different functional groups of various payload classes, such as camptothecins, monomethyl auristatin E, kinesin spindle protein inhibitors (KSPi) and cyclin-dependent kinase 9 inhibitors (CDK-9i). Results: NE-mediated cleavage was found compatible with the diverse linker attachments via hindered ester bonds, amide bonds and sulfoximide bonds. Efficient and traceless release of the respective payloads was demonstrated in biochemical assays. The newly designed SMDCs were highly stable in buffer as well as in rat and human plasma. Cytotoxicity of the SMDCs in cancer cell lines was clearly dependent on NE. IC50 values were in the nanomolar or sub-nanomolar range across several cancer cell lines reaching similar potencies as compared to the respective payloads only in the presence of NE. In vivo pharmacokinetics evaluating SMDC and free payload exposures in rat and particularly the robust efficacy with good tolerability in triple negative breast and small cell lung cancer murine models demonstrate the utility of this approach for selective delivery of payloads to the tumor. Discussion: These results highlight the broad scope of potential payloads and suitable conjugation chemistries paving the way for future SMDCs harnessing the safety features of targeted delivery approaches in combination with NE cleavage in the TME.

Expression of cyclin-dependent kinase 9 is positively correlated with the autophagy level in colon cancer.

Cyclin-dependent kinase 9 (CDK9) expression and autophagy in colorectal cancer (CRC) tissues has not been widely studied. CDK9, a key regulator of transcription, may influence the occurrence and progression of CRC. The expression of autophagy-related genes BECN1 and drug resistance factor ABCG2 may also play a role in CRC. Under normal physiological conditions, autophagy can inhibit tumorigenesis, but once a tumor forms, autophagy may promote tumor growth. Therefore, understanding the relationship between autophagy and cancer, particularly how autophagy promotes tumor growth after its formation, is a key motivation for this research. To investigate the relationship between CDK9 expression and autophagy in CRC, assess differences in autophagy between left and right colon cancer, and analyze the associations of autophagy-related genes with clinical features and prognosis. We collected tumor tissues and paracarcinoma tissues from colon cancer patients with liver metastasis to observe the level of autophagy in tissues with high levels of CDK9 and low levels of CDK9. We also collected primary tissue from left and right colon cancer patients with liver metastasis to compare the autophagy levels and the expression of BECN1 and ABCG2 in the tumor and paracarcinoma tissues. The incidence of autophagy and the expression of BECN1 and ABCG2 were different in left and right colon cancer, and autophagy might be involved in the occurrence of chemotherapy resistance. Further analysis of the relationship between the expression of autophagy-related genes CDK9, ABCG2, and BECN1 and the clinical features and prognosis of colorectal cancer showed that the high expression of CDK9 indicated a poor prognosis in colorectal cancer. This study laid the foundation for further research on the combination of CDK9 inhibitors and autophagy inhibitors in the treatment of patients with CRC.