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Purpose:Bromodomain and extraterminal domain (BET) proteins regulate the expression of many cancer-associated genes and pathways; BET inhibitors have demonstrated activity in diverse models of hematologic and solid tumors. We report the preclinical characterization of INCB054329, a structurally distinct BET inhibitor that has been investigated in phase I clinical trials.Experimental Design:We used multiple myeloma models to investigate vulnerabilities created by INCB054329 treatment that could inform rational combinations.Results:In addition to c-MYC, INCB054329 decreased expression of oncogenes FGFR3 and NSD2/MMSET/WHSC1, which are deregulated in t(4;14)-rearranged cell lines. The profound suppression of FGFR3 sensitized the t(4;14)-positive cell line OPM-2 to combined treatment with a fibroblast growth factor receptor inhibitor in vivo. In addition, we show that BET inhibition across multiple myeloma cell lines resulted in suppressed interleukin (IL)-6 Janus kinase–signal transducers and activators of transcription (JAK–STAT) signaling. INCB054329 displaced binding of BRD4 to the promoter of IL6 receptor (IL6R) leading to reduced levels of IL6R and diminished signaling through STAT3. Combination with JAK inhibitors (ruxolitinib or itacitinib) further reduced JAK–STAT signaling and synergized to inhibit myeloma cell growth in vitro and in vivo. This combination potentiated tumor growth inhibition in vivo, even in the MM1.S model of myeloma that is not intrinsically sensitive to JAK inhibition alone.Conclusions:Preclinical data reveal insights into vulnerabilities created in myeloma cells by BET protein inhibition and potential strategies that can be leveraged in clinical studies to enhance the activity of INCB054329.

Targeted protein degradation (TPD) is an emerging therapeutic modality with the potential to target previously undruggable targets. However, it has been more challenging to identify orally bioavailable TPD molecules due to the physicochemical properties of the large molecules and narrow structure-activity relationship (SAR) compared to conventional small molecule inhibitors. In the present study, we identified orally active TPD molecules that selectively and potently degrade SMARCA2 protein and induce synthetic lethality in SMARCA4-deficient cancer cells. SMARCA2 (BRM) and SMARCA4 (BRG1) are the two mutually exclusive catalytic core subunits of SWI/SNF complexes that play an important role in controlling gene expression by remodeling chromatin. The complexes are mutated in more than 20% of human cancers and subsets of solid tumors lose expression of SMARCA4 protein due to damaging mutations or gene deletion. The SMARCA4-deficient cancer cells are highly dependent on the paralog gene SMARCA2 for their survival and thus SMARCA2 has been suggested as an attractive therapeutic target for patients with SMARCA4-deficient cancers. We have recently identified SMARCA2 selective degraders that demonstrate oral bioavailability in mice with favorable pharmacokinetic properties, and acceptable DMPK and safety profiles in rodent studies. These SMARCA2 degraders show 50 to 300-fold DC50 selectivity for SMARCA2 over SMARCA4 in our cellular assays. When pre-treated with a proteasome inhibitor or neddylation inhibitor, the degradation of SMARCA2 was rescued, confirming that the degradation is mediated by the ubiquitin-proteasome-dependent pathway. These TPD molecules inhibit only SMARCA4-deficient cancer cell proliferation (NCI-H838, NCI-H1693, HT1080 SMARCA4 KO) with IC50 values ranging from 3-10 nM, but not SMARCA4 WT cells (Calu-6, NCI-H520, HT1080 WT). Oral administration of our SMARCA2 degraders resulted in significant tumor growth inhibition of SMARCA4-deficient lung cancer xenografts at well tolerated doses. The treated tumor tissues show robust SMARCA2 protein reduction for more than 72h post dosing, consistent with the SMARCA2 degradation kinetics-based pharmacodynamic prediction model. In summary, our orally bioavailable SMARCA2 degraders induce synthetic lethality in SMARCA4-deficient cancers in vitro and in vivo. Efforts to further evaluate these compounds in additional models and in combination with other agents are ongoing. Citation Format: Koichi Ito, Artem Shvartsbart, Joseph Rager, Anjana Agarwal, Michael Hulse, Komali Vykuntam, Min Wang, Justin Kurian, Miles Cowart, Joy Cote, Monisha Sivakumar, Jack Carter, Jessica Burtell, Alex Grego, Andrew Moore, Neha Bhagwat, Stefan Ruepp, Tom Emme, Liang Lu, Philip Pitis, Corey Basch, Klare Bersch, Song Mei, Raul Leal, John Rose, Danielle Roth, Ganfeng Cao, Kris Vaddi, Sandy Geeganage, Bruce Ruggeri, Andrew Combs, Peggy Scherle. Selective and orally bioavailable SMARCA2 targeted degraders induce synthetic lethality in SMARCA4- deficient solid tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6277.

Cell cycle deregulation is a hallmark of cancer and the hyperactivation and overexpression of CDKs are often drivers of cancer pathogenesis. Cyclin-dependent kinase 4 and 6 (CDK4)/(CDK6) are critical mediators of cellular transition into S phase and important for the initiation, growth, and survival of many cancers. Activated CDK4/CDK6 complexes phosphorylate Rb1, reduce their binding affinities and release Rb1-containing transcription repressor complexes from E2F transcription factors, resulting in activation of E2F controlled cell cycle genes and progression of the cell cycle. At present three CDK4/CDK6 inhibitors are approved for the treatment of ER+/HER2- breast cancer, and are being explored in other cancer indications as well. Previously we described a novel brain penetrant CDK4/CDK6 inhibitor, PRT3645, that exhibits single digit nanomolar biochemical potency against CDK4/CDK6 and >2000-fold selectivity against CDK1, CDK2 and CDK9. PRT3645 inhibits cellular phosphorylation of Rb and exhibits a protein binding-adjusted cellular IC50 of Citation Format: Yue Zou, Srijita Dhar, Kirsten Gallagher, Andrew Buesking, Sarah Pawley, Ryan Holmes, Xiaowei Wu, Katarina Rohlfing, Min Wang, Joseph Rager, Tom Emm, Stefan Ruepp, Miles Cowart, Jing Ni, Jean Zhao, Bruce Ruggeri, Andrew Combs, Kris Vaddi, Sandy Geeganage, Ashish Juvekar, Sang Hyun Lee, Peggy Scherle. The brain penetrant CDK4/6 Inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of NSCLC, CRC, and HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5973.

Adenoid Cystic Carcinoma (ACC) is a rare but aggressive malignancy of salivary gland, associated with protracted clinical course and fatal outcome. Treatment modalities are restricted to surgery and/or adjuvant radiotherapy and patients develop recurrence and distant metastasis over time. The absence of effective systematic therapy makes it incurable in advanced stage. ACC display an overall low mutation frequency, therefore very few actionable genetic alterations critical to the ACC development have been recognized. MYB fusion/overexpression is the most frequently found genetic alteration in ACC and is present in ~70% patients. This is followed by activating NOTCH mutations, reported in ~20% patients. Despite the presence of few targetable genetic alterations, recent studies revealed substantial transcriptomic heterogeneity amongst ACC tumors suggesting the role of epigenetic alterations in ACC oncogenesis. Symmetric dimethylation is an important epigenetic mechanism that regulates mRNA splicing, transcription, translation, cell cycle and oncogenic signaling pathways. Protein Arginine Methyl Transferase 5 (PRMT5) is a predominant enzyme for symmetric dimethylation among a family of 9 methyl transferases. In was suggested that PRMT5 plays a role in regulating tumor progression via modulation of MYC signaling, cancer stemness, and a wide array of additional cellular and transcriptional pro-oncogenic processes. While inhibition of PRMTs showed anti-oncogenic response in several preclinical tumor models and a subset of patients with advanced solid malignancies (including ACC), preclinical studies investigating the effect of PRMT5 blockade in ACC remain inadequate. In part, due to the scarcity of ACC specimens and limited availability of the experimental models. PRT543 is a selective and potent small molecule PRMT5 inhibitor that specifically targets PRMT5 among 37 methyl transferases. In the present study, we have investigated the effect this novel agent using a unique collection of ACC cell lines, organoids, and patient derived xenograft mouse models. To our knowledge, this is the first study investigating the therapeutic effect of PRT543 in several preclinical models of ACC. Specifically, we found that PRT543 has potent antitumor activity in in-vitro and in-vivo models with MYB expression and activating NOTCH mutations. Further, based on these observations, we have sequenced a collection of 50 ACC tumor samples to identify the subset of patients who may potentially benefit from PRT543 treatment based on their underlying genetic signatures. This study provides evidence underscoring the role of PRMT5 signaling in ACC and supports the clinical development of PRMT5 inhibitors for this indication. Citation Format: Vasudha Mishra, Alka Singh, Xiangying Chen, Michael Korzinkin, Claudia Wing, Viktoria Sarkisova, Alexandra Ozerova, Oksana Glushchenko, Venkat Thodima, Koichi Ito, Peggy Scherle, Mark Lingen, Rifat Hasina, Alexander Pearson, Ari Rosenberg, Alex Zhavoronkov, Bruce Ruggeri, Nishant Agrawal, Evgeny Izumchenko. PRT543, a methyl transferase inhibitor, has potent anti-tumor activity against adenoid cystic carcinoma of salivary glands. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4897.

SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core catalytic subunits of the SWI/SNF complexes, containing an ATPase domain and a DNA binding bromodomain. SMARCA4 protein expression is lost in some cancers due to nonsense mutations, and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deleted cancers using selective SMARCA2 degraders induces synthetic lethality while sparing SMARCA4 wild type (WT) normal cells. We have identified PRT3789, a potent and selective SMARCA2 targeted degrader, that selectively inhibits proliferation of SMARCA4-deleted cancer cells. Here, we describe the potential mechanism of action for PRT3789 at the molecular level and the in vitro and in vivo anti-tumor activity in SMARCA4-deleted cancer cells. To further elucidate the SMARCA2 degradation selectivity of PRT3789, we performed mass spectrometry to identify the selective SMARCA2 lysine residues ubiquitinated following treatment with PRT3789. This data, in combination with site-directed mutagenesis against these SMARCA2-specific ubiquitinated residues, has revealed important insights into the mechanism of action of PRT3789. In addition, to further understand the specific vulnerability of SMARCA2 in SMARCA4-deleted cells, we investigated whether PRT3789 affected the integrity of the residual SWI/SNF complex. Coimmunoprecipitation of SMARCC1 revealed that PRT3789 disrupts specific SWI/SNF complex subunits, including ACTL6A (BAF53). Functional genome-wide experiments are ongoing to evaluate the impact of this finding and the residual activity of the SWI/SNF complex. Furthermore, treatment with PRT3789 demonstrated robust inhibition of cell proliferation of SMARCA4-deleted non-small cell lung cancer (NSCLC) cells in vitro and NSCLC PDX tumors ex vivo, but not SMARCA4 WT cancer cells, in a concentration-dependent manner. Lastly, PRT3789 shows favorable pharmacokinetic properties in vivo, which correlate to its pharmacodynamics effects as evidenced by reduced SMARCA2 protein and KRT80 mRNA levels in tumor tissues. In subcutaneous cell-line derived xenograft (CDX) models of NSCLC, administration of PRT3789 demonstrated significant dose-related inhibition of SMARCA4-deleted NSCLC growth at tolerated doses, but no effect on the growth of SMARCA4 WT cancers. In summary, consistent with our previous validation studies and genomic perturbation analyses, our potent and selective SMARCA2 targeted degrader PRT3789 induces strong synthetic lethality in SMARCA4-deleted cancers in vitro and in vivo. Citation Format: Michael Hulse, Anjana Agarwal, Min Wang, Jack Carter, Monisha Sivakumar, Brian Vidal, Justin Brown, Andrew Moore, Alexander Grego, Neha Bhagwat, Joseph Rager, Liang Lu, Corey Basch, Klare Bersch, Chaofeng Dai, Philip Pitis, Andrew Combs, Bruce Ruggeri, Kris Vaddi, Peggy Scherle, Koichi Ito. Preclinical characterization of PRT3789, a potent and selective SMARCA2 targeted degrader [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 3263.

Cell cycle deregulation is a hallmark of cancer and CDK inhibitors, specifically inhibiting CDK4/6 and blocking cells transition from the G1 to the S phase of the cell cycle are the first and only class of highly specific CDK inhibitors approved for cancer treatment to date. CDK4/6 inhibitors have transformed the treatment paradigm of estrogen receptor-positive (ER+), HER2- breast cancer with three CDK4/6 inhibitors currently FDA approved. Brain metastasis commonly arises in patients with breast, lung, melanoma and other cancer types, is associated with poor survival outcomes and poses distinct challenges in clinical management. Due to advances in imaging technologies, the detection of brain metastases is increasing and there is a dearth of novel therapies to combat brain metastatic cancers and impact patient survival. Here, we describe a novel brain penetrant CDK4/6 inhibitor, PRT3645 exhibiting single digit nanomolar biochemical potency against CDK 4/6 and >2000-fold selectivity against other CDK family members (CDK1, CDK2, and CDK9). In cellular assays, PRT3645 inhibits cellular phosphorylation of RB with low nanomolar activity. Consistent with this, PRT3645 treatment resulted in concentration-dependent inhibition of cell proliferation in glioblastoma (GBM) cell lines and in HER2- and HER2+ breast cancer lines (EC50 values < 125 nM). Furthermore, PRT3645 demonstrated additive in vitro activity with fulvestrant or tucatinib in ER+ and HER2+ breast cancer lines. PRT3645 exhibits favorable in vitro safety pharmacology and ADME profiles, including brain exposure in rodents at steady state, and demonstrates oral bioavailability across rodents, dog and nonhuman primates. In vivo, oral PRT3645 was well tolerated and highly efficacious in a dose-dependent manner in subcutaneous xenograft models of GBM and breast cancer and in orthotopic human breast cancer brain metastasis (BCBM) and GBM models in mice as a monotherapy. PRT3645 showed tumor regression as single agent in the MCF7 ER+ breast cancer model and a combinatorial benefit with the estrogen receptor blocker, fulvestrant. In a HER2+ BT474-luc orthotopic model, similarly efficacious single agent activity of PRT3645 was achieved, as well as a significant combinatorial benefit on tumor growth and median survival when administered with the brain penetrant HER2 kinase inhibitor, tucatinib. PRT3645 was highly efficacious in a U87-luc GBM orthotopic model and demonstrated enhanced median survival benefit when combined with an orally active brain penetrant PRMT5 inhibitor. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species, brain penetrance and favorable tissue distribution relative to brain exposure, and currently has advanced into IND-enabling preclinical studies. Citation Format: Ashish Juvekar, Yang Zhang, Andrew Buesking, Min Wang, Dave Rominger, Joseph Rager, Stefan Ruepp, Kirsten Gallagher, Yue Zou, Miles Cowart, Xiaowei Wu, Sarah Pawley, Ryan Holmes, William Gowen-MacDonald, Kris Vaddi, Andrew Combs, Bruce Ruggeri, Peggy Scherle. Brain penetrant CDK4/6 inhibitor PRT3645 demonstrates anti-tumor activity and enhances survival in glioblastoma and breast cancer brain metastasis models [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 2300.

PRMT5 (protein arginine methyltransferase 5) is a major Type II PRMT, which catalyzes the symmetric dimethylation of protein arginine residues (sDMA). As an epigenetic regulator, PRMT5 plays essential roles in promoting cancer growth and survival, including through mechanisms that control alternative splicing and RNA processing, and the expression of DNA damage repair genes. Spliceosome mutations have been suggested to represent a potential biomarker for PRMT5 inhibitors, and our previous work highlighted this sensitivity in SF3B1R625C/G expressing uveal melanoma cells. Here, we highlight in vitro and in vivo activity of PRT543, a potent, selective, and orally available PRMT5 inhibitor, in cancer cells harboring mutations in other spliceosome factors such as U2 small nuclear RNA auxiliary factor 1 (U2AF1) and RNA binding motif protein 10 (RBM10). Mutations in U2AF1 (including S34F hotspot mutations) and RBM10 (primarily loss of function (LOF) mutations) occur in 5-10% of all non-small cell lung cancers (NSCLC). Cell proliferation (10-day assay) was assessed in a panel of NSCLC cell lines treated with PRT543, either wild-type or harboring U2AF1S34F or RBM10LOF mutations. Strikingly, both U2AF1S34F and RBM10LOF cell lines were significantly more sensitive to PRT543 compared to wild-type cell lines. Furthermore, PRT543 induced significant dose-related tumor growth inhibition at well-tolerated doses in cell-line derived xenograft (CDX) models harboring the U2AF1S34F or RBM10LOF mutation. Consistent with our previous findings in other tumor types, PRT543 decreased expression of DNA damage repair-associated genes (e.g. BRCA1, RAD51AP1, FANCA, and FANCL) in U2AF1S34F or RBM10LOF mutant NSCLC cells. Combination with PRT543 increased the effectiveness of specific chemotherapeutic agents in both in vitro and in vivo (CDX) models of U2AF1S34F and RBM10LOF NSCLC. Efficacy studies in patient-derived xenograft (PDX) models, as well as genomic profiling of spliceosome-mutant cellular models in response to PRT543 are ongoing. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Jack Carter, Koichi Ito, Venkat Thodima, Monisha Sivakumar, Michael Hulse, Joseph Rager, Komali Vykuntam, Neha Bhagwat, Kris Vaddi, Bruce Ruggeri, Peggy Scherle. PRMT5 inhibitor PRT543 displays potent antitumor activity in U2AF1S34F and RBM10LOF spliceosome-mutant non-small cell lung cancer in vitro and in vivo [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 2159.

MCL1 is a member of the anti-apoptotic BCL2 family of proteins and plays a critical role in maintaining cellular homeostasis and promoting cell survival. MCL1 amplifications occur frequently in multiple tumor types. It has also been implicated in mediating resistance to chemotherapeutic agents and targeted therapies. We have previously described a novel, potent and orally bioavailable MCL1 inhibitor, PRT1419, that demonstrates anti-tumor efficacy in various preclinical models of cancer and is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies and advanced solid tumors. In an effort to identify novel biomarkers that might predict sensitivity to MCL1 inhibition, we conducted a gene dependency analysis using publicly available human cancer cell line data generated from genome-wide CRISPR/Cas9-mediated cell viability screens. We observed that mutations in the SWI/SNF complex, particularly in lung and ovarian cancer cell lines, conferred a strong functional dependency on MCL1. The mammalian SWI/SNF complex functions as a tumor suppressor in a number of cancers and regulates gene expression via chromatin-remodeling. It is comprised of multiple subunits, including one of two catalytic ATPases (SMARCA2 or SMARAC4), DNA-binding proteins ARID1A, ARID1B and ARID2, and other chromatin-binding subunits. Gene mutations in members of this complex occur in >20% of human cancers, and therapeutic agents targeting its function are under active clinical investigation. We and others have shown potent synthetic lethality with the use of SMARCA2 targeted protein degraders in SMARCA4 deleted lung cancer models. A previously published genome-wide CRISPR screen in SMARCA4-mut lung cancer cell lines demonstrated that loss of MCL1 could sensitize these cells to SMARCA2 degradation. Therefore, we evaluated PRT1419 in combination with a novel and selective SMARCA2 degrader, PRT3789, in SMARCA4 deleted lung cancer models. We observed a potent synergistic interaction in SMARCA4 deleted cell lines in vitro, whereas no additive benefit was seen in SMARCA4 WT lines. Further, combining PRT1419 and PRT3789 in vivo in cell line-derived xenograft models resulted in significant tumor growth inhibition, including tumor regressions. Additionally, we profiled PRT1419 ex vivo in a panel of lung cancer PDX models and observed significant, dose-dependent effects on cell viability in SMARCA4 deleted models with low SMARCA2 expression. In a broader lung cancer cell line viability screen conducted with PRT1419, we observed that the presence of multiple, co-occurring alterations in SWI/SNF family members such as SMARCA4, ARID1A/B mutations and loss of SMARCA2 protein were associated with sensitivity to PRT1419. Based on these findings, preclinical evaluation of PRT1419 in other tumor types with recurrent SWI/SNF mutations is ongoing. Citation Format: Norman Fultang, Neha Bhagwat, Diane Heiser, Alexander Grego, Michael Hulse, Venkat Thodima, Koichi Ito, Kris Vaddi, Bruce Ruggeri, Peggy Scherle. Combination of the MCL1 inhibitor PRT1419 and SMARCA2 degrader PRT3789 shows combinatorial benefit in SMARCA4 deleted lung cancer [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 420.

CDK9 is a serine/threonine kinase belonging to the subclass of the transcription associated CDKs. CDK9 complexes with cyclin T and cyclin K, the positive transcription elongation factor b (P-TEFb), and phosphorylates Serine 2 of RNA polymerase II (pSer2 RNAPII) to activate transcription. Consequently, targeting CDK9 could effectively interfere with epigenetic and transcriptional reprogramming and prevent disease progression and treatment resistance in human cancers. Androgen receptor (AR)-dependence in prostate cancer is linked to CDK9 function. CDK9 stabilizes AR-associated proteins, and pharmacological inhibition of CDK9 can inhibit AR, AR variants, and their downstream transcription programs. We evaluated the novel CDK9 inhibitor, PRT2527, in prostate cancer models to evaluate its effects on cell proliferation, stem-like tumor cells, and tumor growth. PRT2527 is a potent inhibitor of CDK9/CyclinT1 complex, and when evaluated at concentration 200 times the biochemical IC50, PRT2527 was highly selective for CDK9 inhibition. We verified in biochemical assays the ability of PRT2527 to suppress pSer2 RNAPII and reduce expression of c-Myc, a common target of CDK9, in a concentration-dependent manner in multiple human prostate cancer cell lines. PRT2527 also inhibited c-Myc-dependent transcription in vitro in luciferase reporter assays. Furthermore, RNA sequencing showed altered expression of several genes with significant enrichment of c-Myc and E2F targets and RNAPII dependent transcription among downregulated genes in PRT2527-treated VCaP cells. In vitro, PRT2527 inhibited the proliferation of androgen-dependent and androgen-independent prostate cancer cell lines (IC50, ≤50 nM). PRT2527 was highly effective (IC50, ≤10 nM) in tumor-spheroid assays in blocking the growth of stem-like tumor cells and significantly suppressed the in vitro growth of tumor organoids from both human cell lines and patient-derived xenografts (PDXs). In mice, PRT2527 IV administration reduced pSer2 RNAPII in tumor xenografts and c-Myc-dependent transcriptional activity in a DU145 luminescence reporter model. PRT2527 administration in mice significantly reduced growth of PDX LuCaP 35 (castration-sensitive, adenocarcinoma) and LuCaP 145.2 (castration-resistant, neuroendocrine) along with the fraction of tumor-initiating stem-like cells in ex vivo assays. PRT2527 reduced pSer2 RNAPII in both PDXs, whereas c-Myc decreased in LuCaP 35 and Sox2 in LuCaP 145.2, relative to basal expression levels. Collectively, our data demonstrate that PRT2527 has potent pharmacodynamic and antitumor activity in multiple models of castration-sensitive and castration-resistant prostate cancer. PRT2527 is advancing into phase 1 studies in solid tumors. Citation Format: Elisa Federici, Gianluca Civenni, Aleksandra Kokanovic, Giada Sandrini, Luca Guarrera, Simone Mosole, Alessia Cacciatore, Valeria Uboldi, Manuel Lessi, Giovanni Papa, Domenico Albino, Elisa Storelli, Jessica Merulla, Andrea Rinaldi, Marco Bolis, Yang Zhang, Kris Vaddi, Peggy Scherle, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer [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 5471.

Cyclin-dependent kinase 9 (CDK9) is a master regulator of transcription that controls paused RNA polymerase II (RNAP2) release through phosphorylation of its carboxy-terminal domain, resulting in productive transcription elongation. CDK9 has been extensively studied as a potential target for cancer therapy in “transcriptionally addicted” tumors as transient inhibition of CDK9 primarily depletes proteins with short half-lives, such as the oncogenes MCL1 and MYC, making CDK9 a promising target in cancer. Here we show that PRT2527 is a potent and highly selective CDK9 inhibitor with moderate to high clearance that achieves optimal temporal target engagement and exhibits potent in vitro and in vivo activities. PRT2527 inhibited CDK9 enzymatic activity with an IC50 of 0.98 nM in a biochemical assay and showed high selectivity in a panel of kinases when tested at physiologically relevant 1 mM ATP concentration. In vitro, PRT2527 inhibited phosphorylation of Ser2RNAP2 in NCI-H929 cells with an IC50 of 54 nM, and an IC50 of 198 nM in a plasma assay to adjust for human plasma protein binding. Transient treatment of cells with PRT2527 inhibited pSer2RNAP2, depleted MCL1 and MYC proteins, and activated cleaved caspase-3 (CC3) in a concentration-dependent manner. In a proteomic profiling study, MCL1 was identified as one of the major down-regulated proteins following PRT2527 treatment. In a panel of hematological cancer cell lines representing B- and T-ALL, AML, and non-Hodgkin’s lymphoma (NHL), as well as subsets of sarcoma, prostate, adenoid cystic carcinoma (ACC), and non-small cell lung cancer (NSCLC) cell lines, PRT2527 treatment consistently led to a potent, concentration-dependent inhibition of proliferation. In a pharmacokinetic/pharmacodynamic (PK/PD) study, intravenous (IV) administration of PRT2527 achieved transient target engagement, depletion of MCL1 and MYC proteins, and induction of apoptosis in tumor tissue. This PK/PD correlation was successfully translated into in vivo efficacy in multiple models. Once weekly dosing of PRT2527 was well-tolerated and significantly inhibited tumor growth in various AML CDX models and induced tumor regressions in double-hit and triple-hit diffuse large B-cell lymphoma (DLBCL) CDX and PDX models carrying the MYC translocation. Combining PRT2527 with venetoclax achieved complete tumor regressions in a venetoclax resistant OCI-AML3 model. PRT2527 demonstrated potent ex vivo activity in PDX models of B-ALL and T-ALL, as well as various solid tumor PDX models with high levels of MYC amplification and overexpression, including pancreatic carcinoma, gastric and gastroesophageal carcinomas, NSCLC, and sarcoma. In vivo efficacy studies with once weekly IV administration of PRT2527 confirmed significant tumor growth inhibition in select MYC-amplified solid tumor PDX models. Taken together, this preclinical characterization supports the advancement of PRT2527 into clinical studies for transcriptionally addicted hematological malignancies and solid tumors with MYC amplification and/or dysregulation. Citation Format: Yang W. Zhang, Liang Lu, Min Wang, Dave Rominger, Stefan Ruepp, Kirsten Gallagher, William Gowen-MacDonald, Chaofeng Dai, Miles Cowart, Andrew Combs, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P237.