Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common form of acute leukemia in children. Despite this, very little improvement in survival rates has been achieved over the past few decades. This is partially due to the heterogeneity of AML and the need for more targeted therapeutics than the traditional cytotoxic chemotherapies that have been a mainstay in therapy for the past 50 years. In the past 20 years, research has been diversifying the approach to treating AML by investigating molecular pathways uniquely relevant to AML cell proliferation and survival. Here we review the development of novel therapeutics in targeting apoptosis, receptor tyrosine kinase (RTK) signaling, hedgehog (HH) pathway, mitochondrial function, DNA repair, and c-Myc signaling. There has been an impressive effort into better understanding the diversity of AML cell characteristics and here we highlight important preclinical studies that have supported therapeutic development and continue to promote new ways to target AML cells. In addition, we describe clinical investigations that have led to FDA approval of new targeted AML therapies and ongoing clinical trials of novel therapies targeting AML survival pathways. We also describe the complexity of targeting leukemia stem cells (LSCs) as an approach to addressing relapse and remission in AML and targetable pathways that are unique to LSC survival. This comprehensive review details what we currently understand about the signaling pathways that support AML cell survival and the exceptional ways in which we disrupt them.
Venetoclax is a promising agent in the treatment of acute myeloid leukemia, though its antileukemic activity is limited to combination therapies. Mcl-1 downregulation, Bim upregulation, and DNA damage have been identified as potential ways to enhance venetoclax activity. In this study, we combine venetoclax with the dual PI3K and histone deacetylase inhibitor CUDC-907, which can downregulate Mcl-1, upregulate Bim, and induce DNA damage, as well as downregulate c-Myc. We establish that CUDC-907 and venetoclax synergistically induce apoptosis in acute myeloid leukemia cell lines and primary acute myeloid leukemia patient samples ex vivo. CUDC-907 downregulates CHK1, Wee1, RRM1, and c-Myc, which were found to play a role in venetoclax-induced apoptosis. Interestingly, we found that venetoclax treatment enhances CUDC-907-induced DNA damage potentially through inhibition of DNA repair. In vivo results show that CUDC-907 enhances venetoclax efficacy in an acute myeloid leukemia cell line derived xenograft mouse model, supporting the development of CUDC-907 in combination with venetoclax for the treatment of acute myeloid leukemia. The HDAC and PI3K dual inhibitor CUDC-907 synergistically enhances the antileukemic activity of venetoclax in preclinical models of acute myeloid leukemia
Over 40% of acute myeloid leukemia (AML) patients experience relapse after remission from intensive chemotherapy and stem cell transplant. Therefore, novel therapies are in demand to target the root cause of relapse, leukemia stem cells (LSCs). Recently, a selective inhibitor of anti-apoptotic Bcl-2, venetoclax, was FDA approved for treating AML in combination with less intensive therapies for patients with comorbidities. However, anti-apoptotic Mcl-1 overexpression and pro-apoptotic Bim downregulation manifests venetoclax resistance. Our previous work demonstrated that a novel dual inhibitor of PI3K and HDAC, CUDC-907, synergizes with venetoclax by overcoming these resistance mechanisms in bulk AML cells and suppressing c-Myc expression and mTOR activity. Previous literature has reported that Bcl-2 inhibition suppresses oxidative phosphorylation (OXPHOS), which is what LSCs rely on for energy production. This is in contrast to normal HSCs that can readily shift their reliance to glycolysis for ATP production if OXPHOS is inhibited. Since c-Myc and mTOR signaling are also known to support mitochondrial respiration, we hypothesize that the combination of CUDC-907 and venetoclax can selectively target LSCs. Metabolomics analyses revealed that pre-treatment of CUDC-907 followed by venetoclax affects the abundance of intermediates in the TCA cycle of AML cells, prior to apoptosis. RNAseq analyses with the same treatment regimen found that the transcription of the TCA cycle regulator pyruvate dehydrogenase kinase 2 (PDK2) is upregulated, and the transcription of pyruvate carboxylase (PC), aconitase 2 (ACO2) and the glutamine transporter SLC1A5 is downregulated, some of which are known cMyc target genes. These findings were correlated with a reduced oxygen consumption rate, indicating the suppression of OXPHOS. Treatment of patient-derived xenograft (PDX) mice with venetoclax and CUDC-907 reduced primary AML cell engraftment, suggesting both bulk AML cell and LSC suppression. These results suggest that the combination of CUDC-907 with venetoclax may target LSCs by interfering with the TCA cycle. To further establish our hypothesis, C13 glucose flux analysis will assist in following the effected metabolic pathways. Rescue experiments supplementing metabolites that were downregulated in treated AML cells and/or inhibition of PDK2 will confirm the mitochondrial respiration based mechanism of AML cell death by the combination of CUDC-907 and venetoclax. Finally we will confirm that these two drugs when combined, reduce the number of LSCs in primary patient samples with a PDX model and flow cytometry for known LSC markers, CD38, CD34 & CD123. The results of this study will form a solid foundation for the clinical evaluation of this promising combination therapy for the treatment of AML and potentially reduce the incidence of relapse. Citation Format: Katie Hege, Hasini Kalpage, Maik Hüttemann, Holly Pitman, Jeffrey W. Taub, Lisa Polin, Jing Li, Yubin Ge. Enhancing the antileukemic activity of venetoclax against leukemia stem cells by targeting oxidative phosphorylation through dual inhibition of PI3K and HDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2463.
Although standard induction therapy initially elicits a promising response in the majority of acute myeloid leukemia (AML) patients, the majority relapse. Leukemia stem cells (LSCs) that survive chemotherapy are believed to be responsible for AML relapse. Therefore, new therapies that eliminate LSCs are desperately needed. ONC201 is a TRAIL inducer and the founding member of the imipridone family. It has been shown to induce apoptosis in LSCs (Ishizawa et al, Science Signaling. 2016; 9:ra17). ONC201 was chemically modified to increase the potency and selectivity against cancer cells, resulting in the new analog ONC213. In this study, we investigated the antileukemic activity and the underlying molecular mechanism of ONC213 in preclinical AML models. ONC213 activity in AML cell lines and primary AML patient samples was first tested in vitro. MTT assay results revealed that ONC213 IC50s ranged from 91.7 nM to 2.4 µM in AML cell lines and primary AML patient samples, which are achievable in vivo based on results from a PK study in mice (a single dose of 50 and 100 mg/kg ONC213 resulted in peak plasma concentrations of 3.7 μM and 8 μM, respectively). Annexin V/propidium iodide staining and flow cytometry analysis results showed variable responses for the AML cell lines tested. After 48 h treatment with 500 nM ONC213, striking induction of cell death in MOLM-13 and MV4-11 cells was detected (at least 72% Annexin V+ cells), while THP-1 and U937 cells showed little to no increase in Annexin V+ cells (6-11%). Similar results were obtained in primary AML patient samples. In contrast to the 48 h treatment of THP-1 and U937 cells, increasing the treatment duration to 120 h resulted in greater than 50% Annexin V+ cells, suggesting that a longer exposure time is necessary in some cell lines. In MV4-11 and MOLM-13 cells, initiation of cell death was detected 8 to 12 h post ONC213 treatment. Colony formation assays revealed that ONC213 treatment significantly reduced colony formation capacity of primary AML patient samples to less than 5% compared to vehicle control, while having no significant effect on normal hematopoietic progenitor cells. A primary AML patient sample was treated with or without ONC213 for 48 h, transplanted into NSG mice, and ten weeks later bone marrow was harvested and human CD45+ cells were measured. ONC213 treatment significantly reduced human AML engraftment compared to vehicle control (0.6% vs. 21.3%; p<0.05), demonstrating that ONC213 kills LSCs in vitro. Next, we examined in vivo efficacy of ONC213 against an AML cell line derived xenograft mouse model. MV4-11 cells were injected into NSGS mice through the tail vein. Three days post-injection, the mice were randomized into vehicle control or 125 mg/kg ONC213 cohorts (5 mice per cohort) and treated daily for 8 days. Modest weight loss was noted but was entirely manageable. ONC213 treatment extended the survival of mice by 88% (median survival 62 vs 33 days). Unlike ONC201, ONC213 treatment of AML cells did not increase the expression of TRAIL. Interestingly, RNAseq results showed that 500 nM ONC213 treatment for 48 h downregulated 33 mRNAs in the oxidative phosphorylation (OXPHOS) pathway, suggesting that ONC213 treatment decreases OXPHOS in AML cells. Thus far, six of the downregulated mRNAs (UQCRQ, SDHA, COX6C, NDUFS5, ATP5D, and NDUFB1) were verified by real-time RT-PCR after both 8 h and 48 h ONC213 treatment. LSCs have been shown to be highly reliant on OXPHOS, while normal hematopoietic stem cells and some bulk AML cells can switch to glycolysis for ATP production during times of OXPHOS inhibition. Thus, ONC213 may kill LSCs through inhibition of OXPHOS. In addition to downregulation of OXPHOS related genes, we found that ONC213 treatment downregulates Mcl-1. Since Mcl-1 mediates resistance to the promising Bcl-2-selective inhibitor ABT-199 (Venetoclax) and inhibition of Bcl-2 impairs OXPHOS, ONC213 would likely synergize with ABT-199 in AML cells. Indeed, combined treatment resulted in striking synergistic induction of apoptosis in both AML cell lines and primary patient samples. Enhanced cell death was detected 8 h post combination treatment in both MOLM-13 and MV4-11 cells. Results from colony formation assays revealed that the combination spares normal hematopoietic progenitor cells. Taken together, ONC213 is active as a single agent and in combination with ABT-199 in AML. Disclosures Allen: Oncoceutics: Employment. Stogniew:Oncoceutics: Employment. Prabhu:Oncoceutics: Employment. Ge:MEI Pharma: Research Funding.
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