More effective treatment options for elderly acute myeloid leukemia (AML) patients are needed as only 25–50% of patients respond to standard-of-care therapies, response duration is typically short, and disease progression is inevitable even with some novel therapies and ongoing clinical trials. Anti-apoptotic BCL-2 family inhibitors, such as venetoclax, are promising therapies for AML. Nonetheless, resistance is emerging. We demonstrate that venetoclax combined with cyclin-dependent kinase (CDK) inhibitor alvocidib is potently synergistic in venetoclax-sensitive and -resistant AML models in vitro, ex vivo and in vivo. Alvocidib decreased MCL-1, and/or increased pro-apoptotic proteins such as BIM or NOXA, often synergistically with venetoclax. Over-expression of BCL-XL diminished synergy, while knock-down of BIM almost entirely abrogated synergy, demonstrating that the synergistic interaction between alvocidib and venetoclax is primarily dependent on intrinsic apoptosis. CDK9 inhibition predominantly mediated venetoclax sensitization, while CDK4/6 inhibition with palbociclib did not potentiate venetoclax activity. Combined, venetoclax and alvocidib modulate the balance of BCL-2 family proteins through complementary, yet variable mechanisms favoring apoptosis, highlighting this combination as a promising therapy for AML or high-risk MDS with the capacity to overcome intrinsic apoptosis mechanisms of resistance. These results support clinical testing of combined venetoclax and alvocidib for the treatment of AML and advanced MDS.
Hepcidin, a key liver peptide hormone, is essential to the regulation of bioavailable iron and erythropoiesis. Activin-like kinase receptor 2 (ALK2) signaling, via SMAD transcription factors, plays an important part in the regulation of hepcidin expression induced by pro-inflammatory cytokines. In chronic inflammatory conditions, such as rheumatoid arthritis, chronic kidney disease, colitis, and in some forms of cancer, hepcidin expression is induced. This induction of hepcidin expression results in lower levels of bioavailable iron, ultimately leading to the onset of anemia. Hepcidin regulates bioavailable iron levels by binding to and inhibiting the cellular iron pump, ferroportin. Ferroportin is important to macrophage-based iron recycling and dietary iron absorption. Several reports have suggested that lowering hepcidin provides a novel approach for targeting the clinical challenge of anemia. Currently approved approaches for these patients rely on transfusions and the use of erythropoietin-based therapies. Unfortunately, neither of these approaches address the underlying chronic inflammation or hepcidin induction and resulting anemia. In this report, we validate our small molecule inhibitor of ALK2, TP-0184, for the treatment of hepcidin-driven anemia of chronic diseases. Biochemical assays demonstrate that TP-0184 inhibits of the kinase activity of ALK2 with an IC50 of 5 nM. In vitro, TP-0184 is effective at targeting hepcidin expression with an EC50 lower than 100 nM in HepG2 cells. Three in vivo models were also explored for our validation of TP-0184. In our first study, turpentine oil TO was injected into the intrascapular fat pad of C57BL/6 mice to induce an acute inflammatory response that results in hepcidin-driven anemia. The animals were dosed with TP-0184 1 hour prior to TO treatment and once again 8 hours later. The TO-mediated acute inflammatory response in mice resulted in a 14-fold increase in liver hepcidin levels. Two oral doses of TP-0184 at 100 mg/kg, separated by 8 hours, reversed the induction of hepcidin that followed TO treatment. TP-0184 was tested at multiple doses in which efficacy was observed. In our second in vivo model, we induced anemia via intraperitoneal injection with heat-inactivated Brucella abortus. The mice were treated daily with TP-0184 for 3-7 days, after which, whole blood, plasma and livers were collected, from which liver and plasma hepcidin, plasma iron, and complete blood counts were assessed. Treatment with 100 mg/kg TP-0184 completely abrogated the Brucella abortus-induced reduction of hemoglobin and total red blood cell counts. In our third in vivo study, TP-0184 was also evaluated in the TC-1 lung cancer model for cancer-induced anemia. TC-1 tumor-bearing animals exhibited a 3-fold increase in liver hepcidin levels, which was reversed by dosing with 25 mg/kg TP-0184. From these experiments, we conclude that TP-0184 is a potent and selective inhibitor of ALK2 with demonstrated activity in multiple preclinical models of anemia associated with inflammation. TP-0184 also demonstrates favorable pharmacokinetic properties as well as good drug-like qualities, making it a strong candidate molecule with which to move into IND-enabling studies and formal clinical development. The current study supports a clinical development approach focused on anemia of chronic disease where an erythropoietin-sparing approach might offer significant clinical benefit to patients. Disclosures Peterson: Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.
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 system's 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.
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.
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