Wontak Kim

Abstract 235: AXL inhibition leads to a reversal of a mesenchymal phenotype sensitizing cancer cells to targeted agents and immuno-oncology therapies

Mesenchymal properties and the epithelial-to-mesenchymal transition (EMT) contribute to the initiation and progression of many tumor types and ultimately can lead to drug resistance and highly aggressive disease. It is becoming increasingly clear that the more mesenchymal characteristics cancer cells acquire the more resistant they become to standard chemotherapy, targeted agents, and even immune checkpoint inhibitors. We have been exploring the role of the receptor tyrosine kinase, AXL, and its related TAM family members, in promoting the mesenchymal phenotype in cancer cells and how these effects promote drug resistance and escape from immune surveillance. TP-0903, a potent AXL inhibitor, leads to a reversal of the mesenchymal phenotype in multiple cancer models. Following TP-0903 treatment, we observed changes in mRNA expression using RT-qPCR and protein expression using standard immunoblotting that are consistent with a reversal of the mesenchymal phenotype. Upon treatment with TP-0903 cancer cells possessed lower motility and a decrease in anchorage-independent growth, both hallmarks of a mesenchymal cell. In vivo models of erlotinib-resistant non-small cell lung cancer (NSCLC) were utilized to demonstrate TP-0903 single agent activity in highly mesenchymal models; however, more importantly, treatment with TP-0903 was able to sensitize this highly refractory model to erlotinib. AXL function and tumor mesenchymal characteristics also provide mechanisms for the cancer cells to evade immune surveillance. This is achieved by the role that AXL plays in detecting neighboring apoptotic cells resulting in the engulfment of dead cells (efferocytosis) and the associated debris in order to prevent the immune system9s exposure to auto-antigens under normal physiological conditions or exposure to cancer-associated neo-antigens in a tumor. Inhibition of AXL by TP-0903 can potentially inhibit tumor-associated efferocytosis leading to a stronger immunogenic response to the tumor. Indeed, results demonstrated synergy when TP-0903 was combined with an anti-PD-L1 agent in a syngeneic triple negative breast cancer mouse model. Interestingly, during the evaluation of TP-0903 in models of EMT, we detected dramatic change in the expression of the retinoic acid (RA) metabolizing protein CYP26A1, suggesting that AXL inhibition leads to changes in RA metabolism. Our data suggest that AXL induces a transition to a mesenchymal phenotype in cancer cells through the suppression of RA signaling and that TP-0903 can rapidly reverse this phenotype by signaling through RA causing the cell to revert to a more differentiated state. Due to its ability to reverse the aggressive mesenchymal phenotype of cancer cells, TP-0903 is a promising agent with the potential to have single agent activity and combined synergy with targeted anti-cancer agents and immunotherapies. Citation Format: Katherine K. Soh, Wontak Kim, Ye Sol Lee, Peter Peterson, Adam Siddiqui-Jain, Steven L. Warner, David J. Bearss, Clifford J. Whatcott. AXL inhibition leads to a reversal of a mesenchymal phenotype sensitizing cancer cells to targeted agents and immuno-oncology therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 235.

Abstract C202: CDK9 inhibition synergizes with BRD4 inhibitor-mediated super enhancer transcriptional repression in multiple preclinical tumor models

The group of transcriptional regulatory proteins known collectively as the super enhancer complex (SEC) coordinate the expression of entire genetic programs directing cell fate. The SEC is also important in driving cancer progression mediated by transcription of key oncogenes such as c-Myc and Bcl-2. The SEC requires the interaction and coordination of many proteins, including cyclin-dependent kinases (CDK), bromodomain proteins (BRD), histone deacetylases (HDAC), and histone methyltransferases (HMT). Each of these proteins are the focus of significant development efforts for the treatment of cancer. SEC-regulated transcription requires recruitment of CDK9/cyclin T1 from the 7SK RNA/Hexim1 inhibitory complex by BRD4 to transcriptional start sites. CDK9 then phosphorylates RNA polymerase II, releasing it from the start site leading to productive transcriptional elongation and gene expression. Considering the close association of CDK9 and BRD4, we hypothesized that the combination of CDK9 and BRD4 inhibitors would have synergistic effects in cancer cells. Alvocidib is a potent CDK9 inhibitor with validated clinical activity in AML from multiple Phase II studies in over 400 patients. Additionally, BRD4 inhibitors have demonstrated early promise in clinical studies with a focus on hematologic malignancies. However, we have found that CDK9 inhibitors, combined with bromodomain inhibitors, produced a synergistic effect by inhibiting the SEC more effectively than either of these compounds alone. Cell viability studies with various combinations resulted in an increase in potency. This was observed with alvocidib combined with JQ-1 (BRD4 inhibitor) in A549 lung cancer cells. Furthermore, the combination of alvocidib with JQ-1 completely abrogated SEC function, as measured by c-Myc or Mcl-1 expression through RT-qPCR. Similar results were achieved with other combinations of CDK9 and BRD4 inhibitors. These data, primarily focused on alvocidib and JQ-1, suggest a strong rationale for combining CDK9 and BRD4 inhibitors as a treatment strategy for multiple tumor types, including lung cancer. Furthermore, these findings may be more broadly applied to additional therapeutic targets in the SEC. These strategies yield synergistic effects at inhibiting SEC function and are highly active in tumor growth studies of cancer, in vivo. Clinical studies utilizing these combination strategies will explore this therapeutic approach. Citation Format: Ye Sol Lee, Wontak Kim, Katherine K. Soh, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. CDK9 inhibition synergizes with BRD4 inhibitor-mediated super enhancer transcriptional repression in multiple preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C202.

Abstract 1106: Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma

The proteasome inhibitor bortezomib is widely used in the treatment of patients with multiple myeloma (MM). The expression levels of many proteins increase as a result of bortezomib treatment, including the pro-apoptotic protein NOXA. NOXA functions to sequester the anti-apoptotic BCL-2 family member, MCL-1. High levels of MCL-1 and/or low levels of NOXA have been implicated in bortezomib resistance and negative patient outcomes, including short duration of treatment response. The BCL-2-specific BH3 mimetic venetoclax (ABT-199) has also been explored in multiple hematological malignancies, including the treatment of MM. Venetoclax induces apoptosis in a BCL-2 specific manner by directly inhibiting BCL-2 function. However, intrinsic resistance to venetoclax treatment observed in MM patient samples has been attributed to a low BCL-2-to-MCL-1 gene expression ratio, suggesting a central role for MCL-1 in cell survival in this context as well. Increased MCL-1 expression is a known resistance mechanism to venetoclax treatment in a variety of cell types including chronic lymphocytic leukemia and lymphomas. Considering the central role of MCL-1 to treatment efficacy in MM, we investigated the ability of an MCL-1-lowering agent, namely the CDK9 inhibitor alvocidib, to potentiate the activity of venetoclax in MM. Alvocidib suppresses MCL-1 expression via CDK9-mediated regulation of RNA polymerase II. Alvocidib has achieved robust improvements in the clinical response rates of high-risk, newly diagnosed acute myeloid leukemia (AML) patients as part of the time-sequential ACM regimen (alvocidib + cytarabine + mitoxantrone). We therefore hypothesized that alvocidib would potentiate the activity of venetoclax in MM through an MCL-1-dependent mechanism. In this report, we demonstrate that alvocidib inhibits the protein expression of MCL-1 in MM cells in a time-dependent fashion, up to 96 hours. In cell viability assays, the addition of up to 100 nM venetoclax resulted in a 2.8-fold reduction in the IC50 of alvocidib in the cultured OPM-2 cell line. Conversely, the potentiation of venetoclax activity with the addition of alvocidib resulted in a more than 500-fold decrease in IC50 in the relatively venetoclax-resistant OPM-2 cells. Additional studies are currently underway to investigate the efficacy of alvocidib and venetoclax in the context of bortezomib resistance where low NOXA may contribute to enhanced cell survival via MCL-1. Taken together, our data suggest that the combination of alvocidib with venetoclax may constitute a novel therapeutic regimen in the treatment of MM. Further, it suggests that CDK9-mediated targeting of MCL-1 may offer a clinical route to addressing intrinsic resistance in MM patients. Citation Format: Mark Livingston, Wontak Kim, Hillary Haws, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, Steven Weitman, David J. Bearss, Steven L. Warner. Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1106. doi:10.1158/1538-7445.AM2017-1106

Abstract 5133: TP-1287, an oral prodrug of the cyclin-dependent kinase-9 inhibitor alvocidib

Alvocidib is a potent inhibitor of cyclin-dependent kinase-9 (CDK9) and induces apoptosis in cancer cells by reducing the expression of short-lived, anti-apoptotic proteins such as MCL-1. Alvocidib, as a part of a sequential combination regimen with cytarabine and mitoxantrone (ACM), is currently in a Phase II clinical trial in relapsed/refractory acute myeloid leukemia (AML). Patients with AML that have a high dependence on MCL-1 are considered more likely to benefit from the alvocidib-containing regimen. MCL-1 has emerged as a key protein in drug resistance of multiple solid tumor types including breast, prostate and lung cancers. The use of alvocidib in clinical settings beyond the ACM regimen is somewhat limited by the current intravenous route of administration. An orally administered form of alvocidib would allow prolonged repression of MCL-1 through chronic dosing and scheduling. Alvocidib itself is highly permeable in CACO-2 monolayers and is soluble at acidic pHs but solubility is strikingly reduced at neutral or basic conditions, which could hamper the development of an oral formulation. We hypothesized that a phosphate prodrug of alvocidib would improve solubility under neutral or basic conditions and enable the efficient systemic delivery of alvocidib via oral administration. We synthesized a phosphate prodrug of alvocidib, TP-1287, in three steps from the parent compound. The solubility of TP-1287, was determined at various pH levels. It was found to be highly soluble under acidic, neutral, and basic conditions (1.5 mg/mL at pH 2.2; 1.8 mg/mL at pH 4.5; 9.5 mg/mL at pH 6.8 and 9.3 mg/mL at pH 8.7) compared to alvocidib (4.4 mg/mL at pH 2.2; 1.3 mg/mL at pH 4.5; 0.02 mg/mL at pH 6.8 and 0.02 mg/mL at pH 8.7). Pharmacokinetic studies were conducted in mice in which TP-1287 was efficiently converted to the parent alvocidib (Cmax = 1922.7 ng/ml, t1/2 = 4.4 hr) with high oral bioavailability (%F = 182.3, compared to intravenous alvocidib). Efficacy and pharmacodynamic studies (measuring MCL-1 expression levels), were evaluated in tumor xenograft models. TP-1287 demonstrated significant anti-tumor efficacy in the MV4-11 AML mouse xenograft model and produced as much as a 61.7% inhibition of the pharmacodynamic biomarker MCL-1 in xenografted tumors, demonstrating a wide, 75-fold therapeutic dosing window. In addition, TP-1287 strongly inhibited tumor growth, achieving 109.1% tumor growth inhibition (%TGI) at the 7.5 mg/kg dose level. TP-1287 is highly soluble over a broader pH range than alvocidib and is efficiently metabolized to the parent compound in vivo, following oral administration. Tumor xenograft models and pharmacodynamic studies indicate that oral delivery of TP-1287 is efficacious in mice. Based on these results, we anticipate moving TP-1287, as an orally delivered CDK9 inhibitor, into a forthcoming clinical trial directed towards solid tumors vulnerable to the suppression of MCL-1. Citation Format: Wontak Kim, Hillary Haws, Peter Peterson, Clifford J. Whatcott, Steven Weitman, Steven L. Warner, David J. Bearss, Adam Siddiqui-Jain. TP-1287, an oral prodrug of the cyclin-dependent kinase-9 inhibitor alvocidib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5133. doi:10.1158/1538-7445.AM2017-5133

Abstract 3728: Targeting MCL-1 expression, through the inhibition of CDK9 and super enhancer driven transcription, offers multiple opportunities for rational drug combinations

Downregulating the expression and function of MCL-1 through the inhibition of cyclin-dependent kinase-9 (CDK9) has proven to be a valuable strategy to target this important pro-survival signal in malignant cells of numerous cancer types. This is exemplified by the ability of alvocidib, a potent CDK9 inhibitor, to inhibit the expression of MCL-1 at both the transcript and protein levels in multiple cell lines from both hematological and solid tumor origins. The timing and duration of MCL-1 knockdown varies between cell type; however, the knockdown is consistent and in some cell lines persistent after the removal of drug. Although alvocidib has demonstrated single agent activity in both the clinic and in nonclinical models, strategies that exploit MCL-1-dependent drug resistance, are allowing for the more rational use of alvocidib in combination with standard-of-care and investigational agents. Here, we demonstrate that treatment with alvocidib, followed by treatment with cytarabine and mitoxantrone (regimen called FLAM), is synergistic in nonclinical models of acute myeloid leukemia (AML). The FLAM regimen results in a significant increase in apoptosis in comparison to any of the single agents alone. This synergy correlates with the downregulation of MCL-1 expression by alvocidib treatment, which places the cancer cells into a heightened state to undergo apoptosis when induced by cytarabine and mitoxantrone treatments. Additionally, the FLAM regimen has demonstrated robust clinical activity in both front-line and relapsed/refractory AML patients. The knockdown of MCL-1 by alvocidib can also be exploited when used in combination with 5-azacytidine (5-aza). BCL-2 family members, including MCL-1 have been described as mechanisms of resistance to 5-aza. Treatment of cells with alvocidib, to repress MCL-1 expression prior to 5-aza treatment, reduced the 5-aza cell viability EC50 more than 2.5-fold, from 1.8 μM to 0.6 μM in MV4-11 cells. The alvocidib/5-aza combination also resulted in synergistic increases in caspase activity relative to either single agent within the combination, at multiple dose levels. MCL-1 dependence is a known mechanism of resistance to BCL-2-targeting agents, such as venetoclax (ABT-199). Alvocidib is an effective approach to targeting MCL-1 leading to the sensitization of cancer cells to venetoclax. Finally, the rational drug combinations described here are further supported by the finding that MCL-1-dependence, measured by NOXA priming, correlates with clinical benefit from treatment with an alvocidib-containing regimen (eg. FLAM) in AML patients. In conclusion, MCL-1 is a key downstream target of inhibiting CDK-9 with alvocidib. Combination strategies using alvocidib have emerged as a powerful solution for overcoming MCL-1 dependent drug resistance. Citation Format: Wontak Kim, Katherine K. Soh, Ye Sol Lee, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, Steven Weitman, David J. Bearss, Steven L. Warner. Targeting MCL-1 expression, through the inhibition of CDK9 and super enhancer driven transcription, offers multiple opportunities for rational drug combinations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3728.

Abstract 2698: Combination strategies to target super enhancer transcriptional activity by CDK9 and BRD4 inhibition in acute myeloid leukemia

The super enhancer complex (SEC) is a group of transcription regulatory proteins that coordinate the expression of genetic programs which determine cell identity and drive disease states, such as cancer. In acute myeloid leukemia (AML), SECs have been shown to turn on transcriptional programs that drive tumorigenesis and disease progression. The SEC is replete with potential therapeutic targets that have been the focus of many drug development efforts; including cyclin-dependent kinases (CDK), bromodomain proteins (BRD), histone deacetylases (HDAC), and histone methyltransferases (HMT). SEC-regulated transcription begins as CDK9/cyclin T1 is recruited from an inhibitory complex by BRD4 and brought to the transcriptional start site of genes. CDK9 phosphorylates RNA polymerase II, releasing it from the SEC and leading to transcriptional elongation and gene expression. Considering the close association of CDK9 and BRD4, we hypothesized that the combination of CDK9 and BRD4 inhibitors would have synergistic effects, particularly in AML, a disease largely driven by SEC function. Alvocidib is a potent CDK9 inhibitor with validated clinical activity in AML from multiple Phase II studies in over 400 patients. Additionally, BRD4 inhibitors have demonstrated early promise in clinical studies with a focus on AML. We found that CDK9 inhibitors combined with bromodomain inhibitors produced a synergistic effect by inhibiting the SEC more effectively than either of these compounds alone. For example, cell viability studies with various combinations resulted in an increase in potency. This was observed with alvocidib combined with JQ-1 (BRD4 inhibitor) in MV4-11 AML cells. Furthermore, the combination of alvocidib with JQ-1 completely abrogated SEC function, as measured by c-myc expression through RT-qPCR. Similar results were achieved with other combinations of CDK9 and BRD4 inhibitors. The alvocidib and JQ-1 combination was also evaluated in an MV4-11 mouse xenograft model. As single agents, alvocidib (2.5 mg/kg) exhibited a 44% tumor growth inhibition and JQ-1 (25 mg/kg) a 1% growth inhibition. When these two doses were combined there was 100% tumor growth inhibition. These data, primarily focused on alvocidib and JQ-1, suggest a strong rational for combining CDK9 and BRD4 inhibitors as a treatment strategy for AML. Furthermore, these findings could be more broadly applied to additional therapeutic targets in the SEC, such as DOT1L and HDACs. These strategies yield synergistic effects at inhibiting SEC function and are highly active in tumor growth studies of AML in vivo. Clinical studies utilizing these combination strategies are the next steps to further explore this approach. Citation Format: Brigham L. Bahr, Kyle S. Maughan, Katherine K. Soh, Jeremiah J. Bearss, Wontak Kim, Peter Peterson, Clifford Whatcott, Adam Siddiqui-Jain, Steve L. Warner, David J. Bearss. Combination strategies to target super enhancer transcriptional activity by CDK9 and BRD4 inhibition in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2698. doi:10.1158/1538-7445.AM2015-2698

Abstract 1119: Utilization of the TGFβ signaling pathway and cell substrate interaction in a novel anti-cancer drug screen

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The TGF-β signaling pathway has been demonstrated to play a multifunctional and complicated role in cancer progression, making it an intriguing target for anti-cancer therapeutics. TGF-β signaling drives proliferation of cancer cells, invasion at the tumor periphery, and is associated with metastatic potential in a number of cancer types. Tumors expressing TGF-β activity are positively correlated with poorer prognosis in patients. Complicating the role of TGF-β signaling in cancer progression is that TGF-β signaling leads to distinct cellular responses at different stages of cancer progression. How the same signal transduction pathway drives distinct cellular responses is a critical question. One likely possibility is that TGF-β signaling is highly context dependent. Changes in the tumor microenvironment, as occur during cancer progression, alter the outcome of TGF-β signaling. As an extreme example of how TGF-β signaling is affected by micro-environmental stimuli, we and others previously reported that certain non-cancerous cells on rigid collagen I substrates become sensitized to HGF-induced epithelial scattering in response to TGF-β, while cells on pliant collagen I substrates undergo apoptosis in response to TGF-β. Given that tumors are typically rigid tissues, we sought to define whether cancer cells would respond to TGF-β by undergoing apoptosis when cultured on pliant substrates. We plated a wide variety of cancer cell lines on highly pliant substrates, treated then with TGF-β, and observed cell viability over time. While a number of cells showed no apoptotic response to TGF-β signaling under any condition, we find that a significant number of cancer cells derived from solid tumors respond to TGF-β signaling on pliant substrates by undergoing apoptosis. Another group of cell lines undergo apoptosis on pliant substrates in the absence of any TGF-β stimulation. Reasoning that perturbation of cellular signaling that communicated substrate rigidity to cells might sensitize them to apoptosis in response to TGF-β, we designed a high-content high throughput screen to look for molecules that would induce cells to undergo apoptosis in response to TGF-β signaling. This chemical genetics approach will yield cues into the mechanism of tumor microenvironment sensing, as well as provide lead compounds for development of novel therapeutics Note: This abstract was not presented at the meeting. Citation Format: Wontak Kim, Jacob P. Hoj, Nicholas E. Saguibo, Kendra E. Fullmer. Utilization of the TGFβ signaling pathway and cell substrate interaction in a novel anti-cancer drug screen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1119. doi:10.1158/1538-7445.AM2014-1119