Background: Acute myeloid leukemia (AML) is the most common type of adult leukemia. Several studies have demonstrated that oncogenesis in AML is enhanced by kinase signaling pathways such as Src family kinases (SFK) including Src and Lyn, spleen tyrosine kinase (SYK), and bruton's tyrosine kinase (BTK). Recently, the multi-kinase inhibitor ArQule 531 (ARQ 531) has demonstrated potent inhibition of SFK and BTK that translated to improved preclinical in vivo activity as compared with the irreversible BTK inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) models. Given the superior activity of ARQ 531 in CLL, and recognition that this molecule has a broad kinase inhibition profile, we pursued its application in pre-clinical models of AML. Methods: The potency of ARQ 531 was examined in vitro using FLT3 wild type and mutated (ITD) AML cell lines and primary samples. The modulation of pro-survival kinases following ARQ 531 treatment was determined using AML cell lines. The effect of SYK expression on ARQ 531 potency was evaluated using a SYK overexpressing cell line (Ba/F3 murine cells) constitutively expressing FLT3-ITD. Finally, the in vivo activity of ARQ 531 was evaluated using MOLM-13 disseminated xenograft model. Results: Our data demonstrate that ARQ 531 treatment has anti-proliferative activity in vitro and impairs colony formation in AML cell lines and primary AML cells independent of the presence of a FLT3 ITD mutation. We demonstrate decreased phosphorylation of oncogenic kinases targeted by ARQ 531, including SFK (Tyr416), BTK, and fms-related tyrosine kinase 3 (FLT3), ultimately leading to changes in downstream targets including SYK, STAT5a, and ERK1/2. Based upon in vitro drug synergy data, we examined ARQ 531 in the MOLM-13 AML xenograft model alone and in combination with venetoclax. Despite ARQ 531 having a less favorable pharmacokinetics profile in rodents, we demonstrate modest single agent in vivo activity and synergy with venetoclax. Conclusions: Our data support consideration of the application of ARQ 531 in combination trials for AML targeting higher drug concentrations in vivo.
B-cell receptor (BCR) antagonists such as the BTK inhibitor ibrutinib have proven to effectively target chronic lymphocytic leukemia (CLL) tumor cells, leading to impressive response rates in these patients. However patients do still relapse on ibrutinib, and the progressive disease is often quite aggressive requiring immediate treatment. Several strategies are being pursued to treat patients who relapse on ibrutinib therapy. As the most common form of relapse is the development of a mutant form of BTK which limits ibrutinib binding, agents which lead to degradation of the BTK protein are a promising strategy. Our study explores the efficacy of the Hsp90 inhibitor, SNX-5422, in CLL. The SNX Hsp90 inhibitor was effective in primary CLL cells, as well as B-cell lines expressing either BTK wild type or C481 mutant BTK, which has been identified as the primary resistance mechanism to ibrutinib in CLL patients. Furthermore the combination of SNX-5422 and ibrutinib provided a remarkable in vivo survival benefit in the Eμ-TCL1 mouse model of CLL compared to the vehicle or single agent groups (51 day median survival in the vehicle and ibrutinib groups versus 100 day median survival in the combination). We report here preclinical data suggesting that the Hsp90 inhibitor SNX-5422, which has been pursued in clinical trials in both solid tumor and hematological malignancies, is a potential therapy for ibrutinib resistant CLL.
Enhancer of zeste homolog 2 (EZH2) is a part of the polycomb repressive complex and catalyzes the trimethylation of lysine 27 on histone H3 (H3K27me3). EZH2 inhibition has a complex role in the pathogenesis of acute myeloid leukemia (AML), in that it has been shown to be either a tumor suppressor or an oncogene depending on the stage of AML development and the genes that EZH2 is regulating during each stage. Unlike follicular and diffuse large B-cell lymphoma where EZH2 mutations result in gain of function, EZH2 mutations are typically loss of function in myeloid diseases. However, we hypothesized that in AML patients without EZH2 mutations, loss of EZH2 function may produce a phenotype that would allow for therapeutic targeting without influencing normal hematopoiesis. We used EPZ011989 (EPZ), an EZH2 inhibitor tool compound, to inhibit H3K27me3 in our studies. We started by treating the MOLM-13 AML cell line with EPZ and confirmed a decrease in H3K27me3. This reduction in H3K27me3 resulted in a slight decrease in metabolic activity via MTS assays as well as decreased colony formation in methocult. These studies were followed up with EPZ inhibition in primary AML samples in vitro. We found that EZH2 inhibition resulted in decreased self-renewal of primary AML samples but not of CD34+ bone marrow cells from normal donors. Furthermore, we found that after 7-day treatment with EPZ, primary AML samples undergo moderate differentiation as suggested by an increase in CD11b surface expression via flow cytometry. These results are further supported by the morphological changes seen after 14-days of EPZ treatment in vitro. Based on these results, we hypothesize that EZH2 inhibition in primary AML samples promotes the differentiation of AML blasts. Furthermore, our preliminary data suggests that daily treatment with 150 mg/kg of EPZ results in a survival advantage and reduced disease burden in the MOLM-13-luciferase murine xenograft model. Despite loss of function EZH2 mutations portending poor outcomes in myeloid malignancies, we demonstrate that pharmacologic EZH2 inhibition reduces AML blast stemness and promotes differentiation into mature myeloid cells. In contrast, no change in normal CD34+ stem cells occurs with EZH2 inhibition, offering the opportunity to selectively target myeloid leukemia. Citation Format: Sydney Fobare, Ola A. Elgamal, Emily H. Stahl, Abeera Mehmood, Jean Truxall, Mariah L. Johnson, Amina Abdul-Aziz, John C. Byrd, Erin Hertlein. EZH2 inhibition induces blast differentiation in acute myeloid leukemia [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 1824.
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