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.
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
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.
Individually functionalized cation‐ and anion‐based ionic additives are designed to mitigate the interfacial side reaction occurring on both the positive and negative electrode surfaces. By applying 1‐phenyl‐1H‐imidazole‐3‐ium trifluoromethanesulfonate as a surface‐targeting electrolyte additive, the reciprocal failure from multiple electrolyte addition applications is theoretically prevented. Selective interface modification is performed using ionic additives by the migration of cations and anions to the negative and positive electrode surfaces, respectively. A drastic improvement in cycleability compared with that by a general carbonate electrolyte is achieved because the reinforcement of both the graphite and LiNi0.8Co0.1Mn0.1O2 electrode surface is possible by ionic additives. Moreover, a further improvement in the cycle performance compared with that by the typical solid electrolyte interphase‐forming additives, such as vinylene carbonate and fluoroethylene carbonate, is demonstrated by the ionic additive.
Though lithium-ion batteries (LIBs) have seen a meteoric rise in worldwide deployment over the last decade, they should be further advanced in constant demand of higher rate capability and wider temperature adaptability. A solid electrolyte interphase (SEI) is the essential part of LIBs, determining the charge−discharge performance and degradation behavior. Herein, improvement of the SEI properties is achieved by regulating the electrochemical double layer structure with a nonsacrificial electrolyte additive, that is, lithium nonafluoro-1-butanesulfonate. The anion adsorption of the additive affects the decomposition behavior of other additive and solvent species, and the generated SEI at the graphite electrode becomes thinner and more uniform, leading to decreased impedance and finally resulting in improved energy efficiency, power capability, and fast charging performance of the graphite/NCM811 cell. Furthermore, the low-temperature cycleability at −20 °C is considerably enhanced with no dendritic Li metal deposition at the negative electrode surface. A mechanistic study on the interfacial phenomena and the effect is carried out by using various theoretical and experimental methods, such as density functional theory calculations, electrochemical quartz crystal microbalance, and transmission electron microscopy. Consequently, the approach of SEI modification with the nonsacrificial electrolyte additive can be one of the effective ways to advance LIB technology in future.
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.
In individuals with chronic inflammatory diseases, such as cancer or rheumatoid arthritis, constitutive signaling through ALK2, a member of the bone morphogenetic protein (TGFβ/BMP) receptor family, leads to debilitating anemia, commonly referred to as anemia of chronic disease (ACD). Activation of ALK2, like other members of the BMP receptor family, leads to the phosphorylation and activation of SMAD family transcription factors via signal transduction and subsequent activation of gene expression. Activation of ALK2 in the liver induces the SMAD-driven transcription of the peptide hormone hepcidin which, by promoting the degradation of the iron transporter ferroportin, leads to reduced serum iron levels and subsequent functional anemia. Lowering constitutively elevated hepcidin levels by inhibiting ALK2 kinase activity is a potentially viable therapeutic strategy for ACD. Current therapeutic approaches for ACD rely on transfusions, intravenous iron and the use of erythropoietin-based therapies, none of which address the underlying pathological deficit of functionally low iron levels. TP-0184 is a small-molecule, selective inhibitor of ALK2 kinase activity (IC50 = 5 nM). TP-0184 has demonstrated profound preclinical activity in three mouse efficacy models for ACD. In model 1, TP-0184 reversed hepcidin induction in mice treated with turpentine oil. In model 2, TP-0184 abrogated reductions in hemoglobin and total red blood cell counts induced by intraperitoneal injection with heat-inactivated Brucella abortus. In model 3, TP-0184 reversed elevated hepcidin levels in TC-1 tumor bearing mice. Plasma and liver pharmacokinetics in mice revealed that TP-0184 has a high volume of distribution (Vd = 30.8) and accumulates at high concentrations in the liver (Cmax of 292 mM following a single oral dose of at 20 mg/kg). In rat multi-dose tolerability studies, TP-0184 caused no adverse effects when dosed at 200 mg/kg for 7 days, far exceeding the dose levels required to produce efficacy (25 mg/kg). These data suggest that favorable distribution to the liver may play a significant role in the preclinical efficacy of TP-0184 and provide evidence of a significant therapeutic window. Collectively these studies support the clinical evaluation of TP-0184 as an alternative treatment for ACD. Disclosures Peterson: Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.
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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