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Targeting Epigenetic Resistance Mechanisms to PI3 Kinase Inhibition in Leukemic Stem Cells
Shira Glushakow-Smith1,2,5, Imit Kaur1,5, Shayda Hemmati1,5, Ellen Angeles1,5, Taneisha Sinclair1,5, Amit Verma2,3,4,5, Kira Gritsman1,2,3,51Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY,
2Department of Clinical Investigations, Albert Einstein College of Medicine, Bronx, NY,
3Department of Medical Oncology, Montefiore Hospital, Bronx, NY,
4Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY,
5Gottesman Stem Cell Institute, Albert Einstein College of Medicine, Bronx, NY
Acute myeloid leukemia (AML) is a heterogeneous disease associated with poor survival and frequent relapse rates due to the inability to effectively target leukemic stem cells (LSCs), and to acquired resistance to existing treatments. Development of treatments that are able to target LSCs and overcome acquired resistance is crucial to improve the survival of AML patients. The PI3 kinase (PI3K)/AKT signaling pathway is activated in 80% of AML patients, across various AML subtypes, making PI3K an attractive therapeutic target in this disease. Several PI3K inhibitors, such as copanlisib, are clinically approved for other indications with a reasonable toxicity profile.
Several catalytic isoforms of PI3K are expressed in hematopoietic cells. To determine whether these PI3K isoforms play a role in LSC self-renewal, we performed colony assays on KMT2A-AF9 retroviral transduced hematopoietic stem and progenitor cells (HSPCs) with either genetic deletion of PI3K isoforms or pharmacologic inhibition of PI3K isoforms. We found that disruption of PI3K signaling reduces the self-renewal of LSCs and promotes myeloid differentiation in both mouse LSCs and human AML cell lines. The KMT2A-AF9 induces leukemic transformation via aberrant upregulation of key self-renewal genes, such as HoxA9 and Meis1. We found that pharmacologic inhibition of PI3K isoforms leads to a reduction in HoxA9 and Meis1 expression in LSC colonies. In the KMT2A-AF9 retroviral bone marrow transplantation model of AML, genetic deletion of the PI3K isoform Pik3ca leads to a reduction in LSC numbers in the bone marrow and prolonged survival, although it is not curative. We also observed significant prolongation of survival upon secondary transplantation of Pik3ca leukemic cells, suggesting a reduction in functional LSCs. Transcriptome analysis on LSCs isolated from pre-clinical bone marrow aspirates of KMT2A-AF9 primary transplant mice revealed downregulation of the target genes of HoxA9, an important driver of LSC self-renewal. We also observed enrichment of the targets of EZH2, a histone methyltransferase that catalyzes the H3K27 trimethylation repressive mark. AKT is known to phosphorylate EZH2 at Ser21, leading to inhibition of EZH2 function. Therefore, we hypothesized that Pik3ca deletion can promote LSC differentiation by de-repressing EZH2 activation via AKT inactivation.
To determine whether PI3K inactivation differentiates KMT2A-AF9 leukemic cells via dephosphorylation of EZH2, we performed Western analysis on bone marrow from Pik3ca KO and WT leukemic mice. As expected, Pik3ca deletion significantly reduced AKT phosphorylation, leading to a reduction in p-EZH2 levels. Unexpectedly, we also observed reduced levels of total EZH2 protein in Pik3ca KO leukemic cells. We also have preliminary data suggesting a decrease in EZH2 protein levels in AML cell lines after extended PI3K inhibitor treatment, suggesting that EZH2 downregulation may be a non-genetic acquired resistance mechanism to PI3K inactivation. EZH1, a homolog of EZH2, has been reported to compensate for EZH2 function. Thus, we hypothesize that PI3K inhibition may sensitize leukemic cells to EZH1/2 dual inhibition by reducing EZH2 levels. We have data supporting that the dual inhibition with the PI3K inhibitor copanlisib and the EZH1/2 inhibitor valemetostat leads to a combinatorial effect on the proliferation of AML cell lines and AML patient samples. Additionally, we found that these compounds induce myeloid differentiation in the KMT2A-AF9 mouse AML model in vivo. Overall, our data suggests that PI3K plays an important role in leukemic stem cells, and that EZH2 downregulation is a non-genetic mechanism of resistance to PI3K inhibition. Additionally, our findings indicate that combining PI3K inhibitors with EZH1/2 dual inhibitors could be a promising therapeutic approach to prevent resistance and prolong survival in AML patients.
This work was funded by R01CA196973, the V Foundation for Cancer Research, the Alexandrine and Alexander L. Sinsheimer Foundation, and by NIH/National Center for Advancing Translational Science (NCATS) Einstein-Montefiore CTSA Grant Number TL1 TR002557.
Speakers
Shira Glushakow-Smith