Abstract: The NLRP3 inflammasome is an intracellular multiprotein complex that plays a crucial role in the innate immune response by mediating the maturation and release of pro-inflammatory cytokines IL-1 beta, and IL-18. The release of these cytokines leads to the downstream release of TNF-alpha, IL-6, which propagate a robust inflammatory response. Dysregulation of the NLRP3 inflammasome pathway has been linked to various inflammatory diseases, suggesting the possibility for novel therapeutic strategies targeting NLRP3 activation. NLRP3 has been linked to regulating hematopoietic stem progenitor cell (HSPCs) development, expansion, release, and mobility. In addition, various hematological disorders such as myelodysplastic syndrome, myeloproliferative neoplasms, acute and chronic leukemias, and graft-versus-host disease (GvHD) have all been shown to involve NLRP3 inflammasome activation. Given its significant role in these conditions, inhibiting the NLRP3 inflammasome may be a novel therapeutic strategy in hematology and hemato-oncology This study utilized an ex-vivo human whole blood assay to investigate the pharmacodynamics of HT-6184, an allosteric Nek7 inhibitor. We used LPS and ATP to prime and activate the NLRP3 inflammasome and examined the effects of pre-treatment with various concentrations of HT-6184 (starting at 1 mM) on the production of critical pro-inflammatory cytokines, such as IL-1b, IL-18, IL-6 and TNF-a. Notably, preincubation with HT-6184 at concentrations as low as 4.1 nM led to a significant reduction in cytokine production (p & lt;0.0001). Specifically, TNF-α and IL-6 were reduced by 38% and 58%, respectively, while IL-1β and IL-18 were decreased by 44% and 53% respectively. These results provide further evidence of the ability of HT-6184 to modulate the activity of the NLRP3 inflammasome in a complex biological milieu. Our study suggests that HT-6184 has significant therapeutic potential in treating a broad spectrum of inflammasome-mediated diseases. In addition, this assay has the potential to be used to measure the pharmacodynamic properties and the efficacy of HT-6184 and other inflammasome inhibitors in future healthy volunteer clinical trials. In conclusion, this study highlights the importance of innovative and physiologically relevant assay systems in identifying and evaluating potential therapeutic agents for inflammatory diseases.

Abstract: Background The NLRP3 inflammasome is a multiprotein complex composed of several proteins, including NLRP3, ASC, Nek-7, and pro-caspase-1. Many immune cell populations express the NLRP3 inflammasome. In the presence of various exogenous stimuli such as lipopolysaccharide (LPS) or extracellular ATP, the NLRP3 inflammasome components assemble and become activated, releasing pro-inflammatory cytokines IL-1β and IL-18. One of the critical steps in inflammasome assembly is the recruitment of ASC and the formation of ASC protein aggregates called specks. These specks are large enough to be measured through microscopy, and the ASC speck count indicates NLRP3 activation. The release of IL-1β and IL-18 and the upregulation of NLRP3 inflammasome component genes have been observed in many inflammatory and hematological disorders, suggesting the NLRP3 pathway is involved in the disease pathogenesis of many diseases. Purpose HT-6184 is a small molecule that blocks the assembly of the NLRP3 inflammasome through allosteric binding to the protein Nek7. Here we demonstrate the ability of HT-6184 to modulate NLRP3 inflammasome formation by measuring ASC specks using a human monocytic THP-1 cell line engineered to express a GFP-labeled ASC protein. Methods THP1-ASC-GFP cells were cultured in RPMI 1640 with 10% heat-inactivated FBS, 100 μg/mL normocin, and 100 μg/mL Pen-Strep. Cells were seeded at 400,000 cells per well in a 96-well plate and left overnight. Cells were then treated with vehicle or LPS for 3 hours. Following LPS stimulation, cells were treated with vehicle or HT-6184 for 2 hours. After incubation, cells were treated with 5 μM Nigericin and imaged in 3-minute intervals for 1 hour. The images were analyzed for specks. Results By treating THP1-ASC-GFP with LPS and nigericin, we induced the formation of ASC specks and visualized them using fluorescent microscopy. Cells treated with 200 nM HT-6184 had significantly fewer ASC specks than the LPS and nigericin-treated control. ASC speck inhibition by HT-6184 was dose-dependent, as inhibition of speck formation decreased with lower doses of HT-6184. Conclusions HT-6184 effectively prevents the formation of ASC specks in THP-1-ASC-GFP cells. Since THP-1 cells are a monocytic cell line, this data suggests our ability to inhibit the NLRP3 pathway in hematological disorders effectively. By preventing inflammasome formation and activation, we expect to be able to reduce disease burden in hematological diseases.

Abstract: Background The NLRP3 inflammasome is associated with the activation of monocytes and plays a crucial role in the innate immune response. The NLRP3 inflammasome is a multi-protein complex expressed primarily in monocytes and other immune cells. When monocytes encounter danger signals, such as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), the NLRP3 inflammasome is assembled, leading to the maturation and secretion of potent pro-inflammatory cytokines, interleukin-1β (IL-1β), and interleukin-18 (IL-18). These cytokines, in turn, induce a robust inflammatory response to clear infections, promote tissue repair, and coordinate adaptive immunity. However, dysregulated NLRP3 inflammasome activation has been associated with various inflammatory disorders, such as autoimmune diseases, metabolic diseases, and neurodegenerative conditions. Purpose This study investigated the effects of a potent, allosteric NLRP3 inflammasome modulator, HT-6184, on cytokine release response from the activation of NLRP3 in human monocytes. Methods Human peripheral blood mononuclear cells (PBMCs) were utilized, and monocytes were isolated using a Miltenyi pan monocyte isolation kit. Subsequently, these isolated monocytes were exposed to different concentrations of HT-6184 (300 nM and 100 nM) before being stimulated with lipopolysaccharide (LPS) at a concentration of 10 ng/mL, a potent activator of the NLRP3 inflammasome. After 24 hours, the secreted cytokines were analyzed at the single-cell level using the Isospark, with data analysis performed using the Isospeak software. Results The results demonstrated that monocytes treated with LPS alone exhibited high levels of pro-inflammatory cytokines, including effector and stimulatory cytokines, and chemoattractive chemokines such as IL-8, MIF, MIP-1b, and TNF-alpha. In contrast, monocytes treated with HT-6184 and LPS showed reduced cytokine secretion, approaching levels observed in the control group. Notably, the HT- 6184-treated group displayed a lower incidence of polyfunctionality (2%) than the LPS-only group (11%), indicating fewer cells capable of simultaneously secreting multiple cytokines. The 3D-TSNE graph analysis revealed that the HT-6184 and LPS-treated groups clustered with the control group, signifying similar cytokine secretion profiles. In contrast, the LPS-only group exhibited a distinct pattern of cytokine secretion. Conclusions These findings indicate that HT-6184 effectively inhibits the release of pro-inflammatory cytokines upon NLRP3 activation in monocytes. Further research should explore the characteristics of HT-6184 and its ability to modulate the inflammasome in diverse in vitro and in vivo models. The modulation of the NLRP3 inflammasome by HT-6184 holds potential therapeutic implications for managing inflammatory disorders associated with dysregulated NLRP3 activation.

Abstract: TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.

Abstract: Introduction/Purpose: The most common leukemia in adults is Acute myeloid leukemia (AML) and the 5-year overall survival (OS) in all AML patients is approximately 24%. The highest of AML deaths in the US alone is among older patients at ages ≥ 65 emphasizing the need for better therapeutics. The older patients have more limited curative options available due to their inability to tolerate aggressive chemotherapy. Identifying ways to boost immunotherapy responses could change the paradigm of AML, a disease still difficult to treat despite several approved targeted immunotherapeutic. The development of effective small molecules would be significant since small molecules not only target immunosuppressive mechanisms like mAbs but can stimulate intracellular pathways where antibodies are unable to permeate. In addition, small molecules can provide optimal pharmacokinetics and pharmacodynamics for oral administration, amenable clinical dosing, and can induce relatively acute antitumor efficacy to avoid systemic immunogenicity, thus providing a superior therapeutic potential. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the AML leads to depletion of nutrients such as functional/natural amino acids. A key meditator of this nutrient stress pathway, the cytoplasmic GCN2 (EIF2AK4) kinase, switches on reduction of amino acids, and this activity results in T-cell inactivation, T-cell death, regulatory T-cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs). We have discovered a series of novel small molecule immunotherapeutic agents (HCI-1046) that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit responses in AML patient samples and cell lines. Experimental Procedures: GCN2 cell-free kinase binding, EIF2α family selectivity, pGCN2 inhibition assays were performed and confirmed its on-target efficacy and potency of lead inhibitor HCI-1046. Additional experiments were conducted including CellTiterGlo, IncuCyte Live Cell Imaging, FACS Flow Cytometry, Cytotoxicity, Western Blot, ELISpot, and ELISA assays. Results and Summary: GCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF4 inhibition data confirmed on-target activities of our lead GCN2 inhibitor HCI-1046. HCI-1046 demonstrated potent activity, with an IC50 of 36 nM in inhibiting GCN2 kinase, and exhibited cellular efficacy with an IC50 of 0.1 to 0.5 μM range. Our preliminary results support the hypothesis that the inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in the AML cancer model in vivo. Our in vivo PK studies on HCI-1046 in rodent species showed excellent PK properties with 55% oral bioavailability, low clearance, and & gt;5.0-hour half-life. Thus, HCI-1046 is nominated as a pre-clinical agent. Additional data regarding the evaluation of the effects of HCI-1046 on the MDSC-suppressive function on T-cells using ELISpot, ELISA assays in AML patient samples, and other mouse model efficacy studies results will be discussed. Citation Format: Zhaoliang Li, Kyle Medley, Dongqing Yan, Kimberly Coffman, Tony Pomicter, David Lum, David J. Bearss, Hariprasad Vankayalapati. Targeting GCN2 kinase-driven stress response inactivation to restore immunity in AML Cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB090.

Abstract: Myelodysplastic syndrome (MDS) is a heterogeneous group of hematopoietic stem cell disorders. Approximately 70% of patients diagnosed with higher-risk (HR) MDS progress to acute myeloid leukemia (AML). Cyclin-dependent kinase 9 (CDK9) influences transcription through phosphorylation and activation of RNA polymerase II (RPB1), which increases the levels of key oncogenic survival genes like myeloid cell leukemia-1 (MCL-1). MCL-1 dependent malignancies can be identified through a functional assay by adding a NOXA mimetic MCL-1 antagonist peptide to patient samples and measuring the subsequent induction of apoptosis. The assay has been tested in bone marrow mononuclear cells (BMMCs) from AML and MDS patients to examine MCL-1 dependency prior to therapy with alvocidib. Approximately 25% of AML patient BMMCs and 60% of MDS patient BMMCs are MCL-1 dependent (data not shown). We hypothesized that the hypomethylating agents (HMAs) azacitidine and decitabine, which are currently approved for treatment of MDS, can increase MCL-1 dependency through re-expression of pro-apoptotic proteins like NOXA, normally repressed through DNA methylation in malignancies. In the MV-4-11 AML cell line and primary CD34+ MDS BMMCs, alvocidib treatment resulted in a dose dependent reduction in p-RPB1 and MCL-1. In cell viability assays using CellTiter-Glo, treatment with alvocidib and azacitidine resulted in IC50 values of ~100 nM and ~3000 nM, respectively in both cell types. Treatment with HMAs increased NOXA expression and alvocidib suppressed MCL-1 expression, whereas sequential treatment of azacitidine and alvocidib showed both NOXA induction and MCL-1 suppression. Sequential treatment of azacitidine and alvocidib showed synergistic apoptosis induction compared with either treatment alone. Azacitidine treatment sensitized MV-4-11 and MDS BMMCs to MCL-1 inhibition in an MCL-1 dependency assay. In an in vivo efficacy study using the MOLM13 AML xenograft model, the combination of alvocidib and azacitidine or decitabine inhibited tumor growth 71% and 84%, respectively. Alvocidib, azacitidine and decitabine alone showed 51%, 5.7% and 19% tumor growth inhibition (TGI), respectively. Pharmacodynamic analysis in a Phase 1 trial with alvocidib (clinicaltrials.gov, NCT03593915) showed DNA methylation of NOXA gene locus was reduced and NOXA gene expression was upregulated, while MCL-1 gene expression was suppressed in peripheral blood mononuclear cells (PBMCs) after azacitidine and alvocidib treatment. These pre-clinical and clinical data suggest that an alvocidib/HMA combination may constitute a viable therapeutic regimen whose rationale focuses on hypertargeting of NOXA/MCL-1. Taken together, these studies indicate that the combination of alvocidib and HMAs drives AML/MDS cells toward MCL-1 dependent apoptosis. Citation Format: Yuta Matsumura, Ethika Tyagi, Satya Pathi, Dan D. Vo, Tianxiang Zhu, Suman Verma, Clifford J. Whatcott, Stephen P. Anthony, Adam Siddiqui, Jason M. Foulks, David J. Bearss, Steven L. Warner. CDK9 inhibition combined with hypomethylating agents target MCL-1 dependency in MDS and AML [abstract] . In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1959.

Abstract: Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFβ signaling and induced JAK1–STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1–STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFβ, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFβ, and JAK1–STAT3 signal activation in select tumors that may be targeted by combined AXL–JAK1 inhibition. Significance: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

Abstract: Background: Alvocidib is an investigational cyclin-dependent kinase-9 (CDK9) inhibitor that can suppress RNA polymerase II-mediated transcription of genes implicated in leukemia cell survival, including myeloid leukemia cell-1 (MCL-1). MCL-1 is an anti-apoptotic BCL-2 family member that is a key mediator of apoptosis in AML. Alvocidib combined in a timed-sequential regimen with cytarabine and mitoxantrone (ACM) has shown clinical activity in newly diagnosed and relapsed/refractory (R/R) AML through Phase I and II clinical trials. Analysis of bone marrow samples from newly diagnosed AML patients (pts) treated with ACM showed an association of complete remission (CR) with MCL-1 dependence by a BH3 profiling biomarker assay. Zella 201 was initiated based on the hypothesis that ACM may have preferential clinical activity in pts with MCL-1 dependence. We report the findings from an exploratory cohort of newly diagnosed high-risk (NDHR) AML pts with MCL-1 dependence treated with ACM. Methods: Zella 201 is a biomarker-driven Phase II study of ACM in R/R AML patients with MCL-1 dependence. Stage 1 included a cohort of R/R AML pts with various levels of MCL-1 dependence and an exploratory cohort of NDHR AML with MCL-1 dependence & gt;40%, as determined by a BH3 profiling assay. Eligibility criteria for the NDHR cohort included pts 18-65 years with high-risk AML defined as one of the following: A) treatment-related AML, B) AML from preexisting MDS/MPN, C) adverse-risk by ELN 2017 criteria. Induction therapy consisted of alvocidib 30 mg/m2 as a 30 minute IV bolus followed by 60 mg/m2 over 4 hours on Days (D) 1-3, cytarabine 667 mg/m2/D by continuous IV infusion D6-8, and mitoxantrone 40 mg/m2 IV on D9. Up to 3 additional cycles of the same regimen (with or without mitoxantrone) were permitted in responders. The primary endpoint was CR/CRi. Key secondary endpoints were overall survival (OS), relapse-free survival (RFS), overall response rate and safety. Results: Thirteen NDHR pts were treated and evaluable in this cohort (Table 1). One pt received alvocidib on days 1-3 and withdrew from the study on day 6 due to grade 4 diarrhea, cytokine release syndrome, and acute kidney injury. This pt was excluded from the efficacy analysis. Median MCL-1 score was 56% (Range: 42-70%). This cohort was influenced by the following poor risk categories: secondary AML (n= 9; 69%), adverse-risk by ELN (n=8; 62%) and TP53 mutations (n=6; 46%). The most common ≥Grade 3 treatment-emergent non-hematologic AEs (n=14) were diarrhea (29%); TLS, hypocalcemia, sepsis, hypotension (21%), pneumonia, colitis, hyperglycemia, anorectal infection, dyspnea, and left ventricular dysfunction (all 14%). Overall, CR/CRi was 62% with 7 (54%) pts responding following 1 cycle of therapy and another pt achieving CR after a second cycle. Two of six pts with TP53 mutation achieved CR. Although all pts included in this cohort were determined to be MCL-1 dependent, there was no association of CR with increasing MCL-1 dependence. Six (46%) pts went on to an allogeneic stem cell transplant (SCT). Sixty-day mortality was 0%. Median follow-up, OS, and RFS were 8.0, 8.5, and 6.1 months, respectively. Five of 8 (68%) CR/CRi pts have relapsed, and 10 pts (77%) have expired to date. The three pts still alive all received a post-study SCT. Conclusion: ACM has clinical activity in a limited cohort of NDHR AML pts with MCL-1 dependence scores & gt;40% in a biomarker assay. Despite observed CR rates, duration of CR was modest and overall outcomes were poor. These results are comparable to historical controls with conventional chemotherapy regimens given the high-risk subset (62% of pts had adverse-risk and 46% had TP53 mutations). Further study is warranted to better define subgroups of ND AML pts who may benefit from alvocidib-containing induction regimens. Disclosures Zeidner: AsystBio Laboratories: Consultancy; AROG: Research Funding; Forty-Seven: Other: Travel Reimbursement, Research Funding; Merck: Research Funding; Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding; Daiichi Sankyo: Honoraria; Genentech: Honoraria; Pfizer: Honoraria; Takeda: Consultancy, Honoraria, Other: Travel Reimbursement, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Other: Independent Review Committee; Agios: Honoraria. Lee:Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding; Novartis: Research Funding; Genentech: Research Funding; Forty Seven: Research Funding; Bayer: Research Funding; AbbVie: Research Funding; Celgene: Consultancy. Fine:Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Wang:Bristol Meyers Squibb (Celgene): Consultancy; Jazz Pharmaceuticals: Consultancy; Abbvie: Consultancy; Pfizer: Speakers Bureau; Genentech: Consultancy; Stemline: Speakers Bureau; PTC Therapeutics: Consultancy; Macrogenics: Consultancy; Astellas: Consultancy. Bhatt:Incyte: Consultancy, Research Funding; Oncoceutics: Other; National Marrow Donor Program: Research Funding; Jazz: Research Funding; Partnership for health analytic research: Consultancy; Takeda: Consultancy; Omeros: Consultancy; Agios: Consultancy; Rigel: Consultancy; Tolero: Research Funding; Pfizer: Research Funding; Abbvie: Consultancy, Research Funding. Kolibaba:Verastem: Honoraria; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Compass Oncology: Ended employment in the past 24 months; Seattle Genetics: Research Funding; Atara Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sumitomo Dainippon Pharma Oncology, Inc.: Consultancy, Other: Travel, Accommodations, Expenses Paid; Genentech: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Celgene: Research Funding; AbbVie: Research Funding; Acerta: Research Funding; McKesson Life Sciences: Consultancy; Cell Therapeutics: Research Funding; Pharmacyclics: Research Funding. Anthony:Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment; Exact Sciences: Consultancy. Bearss:Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Smith:Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract: Cyclin-dependent kinase 9 (CDK9) is a promising target for cancer therapy due to its regulation of RNA polymerase II (RPB1) and key downstream anti-apoptosis proteins such as myeloid cell leukemia-1 (MCL-1). TP-1287 is a novel oral prodrug of alvocidib, a potent CDK9 inhibitor, and is currently under clinical investigation in patients with advanced solid tumors (clinicaltrials.gov, NCT03604783). TP-1287 is converted to alvocidib by alkaline phosphatases. CDK9 mediates phosphorylation of RPB1 at Ser2 and subsequently drives transcription of key oncogenic signaling genes, such as MCL-1. MCL-1 is a critical factor for survival of tumor types including acute myeloid leukemia (AML), multiple myeloma (MM) and myelodysplastic syndromes (MDS). MCL-1 is also expressed in normal peripheral blood mononuclear cells (PBMCs), thus it was hypothesized that CDK9 inhibition can be assessed clinically using PBMCs as a surrogate biomarker at multiple time points. We hypothesized that alvocidib, the pharmaceutically active form of TP-1287, would modulate CDK9 signaling pathways in cancer cells, resulting in tumor growth inhibition. In cell viability assays using CellTiter-Glo to evaluate the in vitro anti-tumor activity of alvocidib, we observed an IC50 of around 100 nM in AML and MM cell lines: 53-78 nM in MOLM-13, MV-4-11, and HL-60 AML cell lines and 31-223 nM in NCI-H929 and RPMI-8226 MM cell lines. In flow cytometry analysis, alvocidib reduced phosphorylation of RPB1 (p-RPB1) and MCL-1 protein expression in RPMI-8226 and MV-4-11 cells in a dose and time-dependent manner. In a pharmacodynamic study using the RPMI-8226 xenograft model after a single oral administration of TP-1287, we observed a substantial decrease of p-RPB1 and MCL-1 in tumor tissues followed by induction of cleaved caspase-3, an indicator of apoptosis. Furthermore, in an in vivo efficacy study using the RPMI-8226 model, TP-1287 achieved tumor growth inhibition (%TGI), ranging from 17.3% at 0.5 mg/kg (QD) to 86.6% at 15 mg/kg (Q7D) at day 22 after treatment. We hypothesized that PBMCs could serve as a surrogate tissue to measure CDK9 inhibition. To assess this approach in PBMCs, we performed ex vivo assays using human PBMCs from multiple healthy donors. Alvocidib demonstrated dose-dependent p-RPB1 and MCL-1 inhibition after 24 hr treatment. Preliminary data from the Phase 1 trial with TP-1287 showed p-RPB1 suppression in PBMCs from multiple patients, suggesting the successful execution of this assay and evidence of target engagement in the clinical setting. Taken together, TP-1287 demonstrated potent cell and tumor growth inhibition in multiple hematological cell lines, including AML and MM. Furthermore, a newly established flow cytometry system for p-RPB1 and MCL-1 to evaluate CDK9 inhibition in human PBMCs was developed, which could be useful as a surrogate biomarker for TP-1287 in clinical trials and warrants further investigation. Citation Format: Yuta Matsumura, Ethika Tyagi, Clifford J. Whatcott, Jason M. Foulks, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. Pharmacodynamic biomarker strategies for CDK9 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5813.

Abstract: Sumitomo Dainippon Pharma Oncology (f/k/a Tolero Pharmaceuticals), Lehi, UTTNK1 is a poorly understood member of the ACK family of non-receptor tyrosine kinases with a unusual domain arrangement, but no known mechanism of regulation nor conclusive link to disease. We initially identified TNK1 as a mediator of cell survival in a subset of primary patient cancer samples. In an effort to understand how TNK1 is regulated, we discovered a MARK-mediated phosphorylation at S502 near the TNK1 C-terminus that mediates an interaction with 14-3-3. We found that 14-3-3 binding inhibitsTNK1 kinase activity, whereas disruption of this interaction renders TNK1 highly active and capable of driving tumor growth in vivo. In support of this idea, kinase substrate profiling at the proteome level revealed a variety of pro-growth and motility substrates for active TNK1. One unique feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus, which sits adjacent to the 14-3-3 binding site. We found that the TNK1 UBA has a high affinity for a variety of poly-ubiquitin linkages—making TNK1the first kinase, to our knowledge, that directly interacts (non-covalently) with ubiquitin. Remarkably, point mutations within the UBA that disrupt ubiquitin binding inhibit TNK1 activation and oncogenic signaling, revealing a unique UBA-centric mechanism of tyrosine kinase regulation, in which TNK1toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we used a structure-guided approach to identify novel small molecule TNK1 inhibitors with high potency and selectivity. One such compound, TP-5809, inhibits TNK1 dependent STAT5 phosphorylation in vitro and in vivo in L540, a Hodgkin lymphoma cell line with a TNK1-activating mutation. TP-5809 also potently inhibits the in vitro growth of mutant TNK1-driven pro-B cells and reduces disease burden and prolongs survival in mice with xenografted tumors. Together, our data elucidate the first mechanism of TNK1regulation and identify lead compounds for the development of a TNK1 inhibitor. Citation Format: Tsz-Yin Chan, Christina Egbert, Logan Larsen, Jeremy Tsang, Julia Maxson, Eranga Roshan, Clifford J. Whatcott, Kim Wontak, Gaelle Mercenne, Savannah Free, Adam Siddiqui, Tetyana Forostyan, Ethika Tyagi, Kenneth A. Christensen, David J. Bearss, James Moody, Jeffrey Tyner, Jason M. Foulks, Steven L. Warner, Joshua Lyon Andersen. Elucidation of a unique regulatory mechanism for TNK1 provides potential therapeutic targeting opportunities in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2307.