MAPK3 modulates enhancer-promoter interactions of SKAP2 in acute myeloid leukemia.

Validation of NMT1 and NMT2 As Novel Drug Targets in Adult Acute Myeloid Leukemia: Rationale for N-Myristoyltransferase Inhibition with Pclx-001 for Clinical Trials

Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. In acute myeloid leukemia (AML), upregulation of Lyn and Src, important myristoylated proteins, contribute to cell survival and proliferation. The specific roles of NMT1 and NMT2 are unknown in this context. The relationships among NMT1/NMT2 expression and acute AML patient outcomes were studied using RNA-sequencing and microarray cohorts with over 350 patients. We found that high NMT1 and low NMT2 expression were associated with reduced overall and event-free survival in adult AML, which was independent of other prognostic markers on multivariate analysis. High NMT1, but not NMT2, expression was associated with proliferative gene sets in AML cell lines, indicating potential for distinct isozyme substrates. Given these results, we examined NMT1 and NMT2 levels in AML cell lines and AML patient blast cells using Western blotting and flow cytometry. We determined that NMT2 expression varied greatly among patients, but was markedly reduced in most cases, while NMT1 expression was relatively preserved. A potent small molecule NMT inhibitor, PCLX-001, preferentially induced apoptosis and reduced viability in NMT2-deficient AML cell lines cultured in vitro compared with normal lymphocytes and peripheral blood mononuclear cells. PCLX-001 also killed freshly isolated human AML blasts ex vivo with an IC50 of ~170nM regardless of their mutational background. In a murine AML xenograft model, subcutaneously delivered PCLX-001 monotherapy demonstrated dose-dependent anticancer activity and produced complete remissions after five daily 50 mg/kg doses. NMT expression provides independent prognostic information to refine existing clinical stratification, and NMT inhibition is a promising novel therapeutic strategy for AML. Citation Format: John R. Mackey, Aishwarya Iyer, Megan C. Yap, Zoulika Zak, Krista Vincent, Erwan Beauchamp, Lynne M. Postovit, Joseph Brandwein, Luc G. Berthiaume. Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3043.

Background: Circular RNAs (circRNAs) are non-coding RNAs generated via back-splicing. They are indirect – yet pivotal – regulators of gene expression, as many of them bind microRNAs (miRNAs) and render them unavailable to exert their direct regulatory functions. They also interact with chromatin, altering its structure and affecting transcription of several genes. Lastly, though considered as non-coding, few of them have also been shown to encode short, functional polypeptides. Due to their multifaceted role and the deregulation of their expression in cancer, circRNAs are related to the development and progression of malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). BCL2L12 is a prominent member of the apoptosis-related BCL2 family, with an already established role in AML. Moreover, BCL2L12 mRNA expression has been associated with chemoresistance of malignant bone marrow blasts. However, the identity and role of the circRNAs produced by alternative back-splicing of BCL2L12 primary transcripts in MDS and AML has not been investigated, so far. Aims: We sought to determine the identity of BCL2L12 circRNAs expressed in MDS and AML cell lines and to explore their putative interactions with miRNAs having an established role in these disease entities. Methods: MDS-L cells [MDS with del(5q) and complex karyotype], NB-4 and HL-60 cells (acute promyelocytic leukemia with and without PML-RARA, respectively), U-937 and THP-1 cells (acute monocytic leukemia), KASUMI-1 cells (AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1) and MOLM-13 cells (AML with t(9;11)(p21.3;q23.3); KMT2A-MLLT3) were propagated. Total RNA was extracted from these seven cell lines; 2μg of each RNA extract were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of the BCL2L12 gene; thus, only cDNAs from BCL2L12 circRNAs were amplified. After having mixed and purified all nested-PCR products of each cell line cDNA, nanopore sequencing libraries were built. Following nanopore sequencing and basecalling in a MinION Mk1C 3rd-generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA identification was performed using an in-house–built, PERL-based algorithm; miRDB was used for prediction of their interactions with miRNAs. Results: We discovered 83 novel BCL2L12 circRNAs, 9 of which were common between MDS-L cells and at least one AML cell line. The exon structure of these novel circRNAs showed a remarkable diversity, comprising exons also encountered in messenger RNAs (mRNAs) of BCL2L12 as intronic regions. This phenomenon was mostly observed in MDS-L cells. Several BCL2L12 circRNAs in NB-4 and HL-60 cells exhibited complete intron retention; additionally, a novel exon was present at a remarkable frequency in NB-4 cells. Moreover, several BCL2L12 circRNAs are predicted to sponge miRNAs with a key role in AML (miR-7-5p, miR-125b-5p and miR-182-5p) and/or MDS (miR-150-5p). Summary/Conclusion: Numerous circRNAs are produced through alternative back-splicing of the primary BCL2L12 transcripts. The circRNA expression profile of this gene differs among the MDS and AML cell lines, as well as among the six AML cell lines. Intronic regions of this apoptosis-related gene are included in several circRNAs, augmenting the repertoire of miRNAs predicted to be bound by BCL2L12 transcripts. This phenomenon was more common in the MDS cell line, followed by both acute promyelocytic leukemia cell lines. Our data suggest an important regulatory role for this gene in MDS and AML subtypes.

Proton Sensor GPR68 Is Essential to Maintain Myeloid Malignancies

PIM Inhibition By SEL24/MEN1703 Combines Synergistically with Gilteritinib in FLT3-ITD Preclinical Models of Acute Myeloid Leukemia

Molecular Interactions between Mitochondrial Ferroptosis and Apoptosis Pathways in Acute Myeloid Leukemia

The Role of pp90rsk-Mediated CREB Phosphorylation in Acute Myelogenous Leukemia

Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK Cell-Mediated Cytotoxicity Against Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with Erwinia-derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the de novo serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR-Cas9-mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC50) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic anti-proliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML.

Alterations in differentiation pathways contribute to the development of acute myeloid leukemia (AML). Differentiation therapy with all-trans retinoic acid (ATRA) has dramatically altered the treatment of acute promyelocytic leukemia, transforming it from a nearly fatal disease to a curable one. We set out to identify cellular pathways that contribute to AML differentiation, with the goal of identifying new therapeutic targets. We analyzed gene expression data from AML cell lines treated with phorbol 12-myristate 13-acetate (PMA), ATRA, Vitamin D, the BET inhibitor JQ1 and the DOT1L inhibitor EPZ00477, treatments known to induce AML differentiation and impair growth. Folate-mediated one-carbon metabolism was one of only three metabolic pathways altered by these compounds, with expression of MTHFD2 consistently downregulated with each compound.MTHFD2 is an NAD-dependent, bi-functional mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase. It is differentially expressed in embryonic and transformed tissues and is upregulated in myeloid progenitors. MTHFD2 is the most differentially expressed metabolic enzyme in cancer cells versus normal cells, including normal proliferating cells. We thus investigated the role of MTHFD2 in myeloid malignancy. First, we demonstrated using ChIP-qPCR, MYC knockdown and MYC inhibition with a BET inhibitor, that MYC directly regulates MTHFD2 expression in AML. Knockdown of MTHFD2 with two shRNAs confirmed to have on-target activity, induced myeloid differentiation in AML cell lines, as measured by Cd11b expression, morphologic changes and induction of a previously validated AML differentiation gene expression signature. MTHFD2 knockdown decreased cell growth in AML cell lines, as well as decreased colony formation in methylcellulose in both AML cell lines and primary patient blasts. AML cells transduced with these two MTHFD2-directed shRNAs demonstrated attenuated growth in an orthotopic mouse model of AML. Furthermore, three MTHFD2-directed shRNAs prolonged survival in an MLL-AF9 mouse leukemia model. Additionally, using a doxycycline inducible shRNA system, we demonstrated that two miR30-shRNAs directed against MTHFD2 decreased AML burden in mice with established disease and prolonged survival.To identify biomarkers of response to MTHFD2 suppression, we used single sample Gene Set Enrichment Analysis (ssGSEA) to identify primary patient AML samples enriched for gene expression signatures of folate-mediated one-carbon metabolism and MTHFD2. We found in both independent data sets that the cluster of patients enriched for expression of the one-carbon folate pathway gene signatures was also enriched for patients with FLT3-ITD mutations, a subset of AML with a particularly poor prognosis. In addition, in an shRNA screen targeting 11,194 genes and performed in 216 cancer cell lines, including 17 AML lines, FLT3-ITD was a biomarker of response to MTHFD2 knockdown. We next validated that while MTHFD2 suppression induced measureable differentiation in all six AML cell lines examined, it induced the most robust induction of apoptosis in FLT3-ITD mutant AML. The mitochondrial one-carbon folate pathway is thought to contribute to cellular oxidative balance by providing reducing power in the form of NAD(P)H, and suppression of MTHFD2 was previously shown to increase ROS levels. Indeed, suppression of MTHFD2 led to a marked increase in ROS in the FLT3-ITD positive AML cell lines in which apoptosis was induced.In summary, a decrement in MTHFD2 expression was found at the center of multiple AML perturbations that impair AML growth and induce differentiation. Our data support MTHFD2 as an AML dependency and FLT3-ITD as a potential biomarker of response. We thus provide critical preclinical evidence for targeting of MTHFD2 as a therapeutic strategy in AML. DisclosuresStone:Celgene: Consultancy; Merck: Consultancy. DeAngelo:Celgene: Consultancy; Pfizer: Consultancy; Incyte: Consultancy; Agios: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Amgen: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.

Loss of CCAAT‐Enhancer Binding Protein Delta Promotes Acute Myeloid Leukemia Cell Proliferation and Survival By Upregulating Cyclin D1 Expression

An Aberrantly Overexpressed Short Isoform of TET1 Lacking a DNA Binding Domain Drives Aberrant 5-Hydroxymethylation Marks, Oncogenic Pathways, and Growth in Acute Myeloid Leukemia

Background: Fatty acid synthase (FASN) upregulation during conditions of oxidative stress contributes to tumor proliferation and survival, which appears to be a mechanism of proteasome inhibitor resistance. Here we demonstrate that acute myeloid leukemia (AML) cell lines resistant to carfilzomib (CFZ), a second-generation proteasome inhibitor, have higher basal FASN expression and targeting FASN with small molecule inhibitors enhances the cytotoxic effect of CFZ in both AML and multiple myeloma (MM) cell lines. Methods: In a proliferation assay, human AML and MM cell lines were treated with a single dose of CFZ for 7 days. Inhibition of proliferation was defined using an IC50 cutoff for CFZ of 10nM for AML and 5nM for MM as the threshold for sensitivity. Sensitive and resistant cell lines were subjected to apoptosis and cell cycle analyses by flow cytometry after being exposed to CFZ for 72 hours. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). For CFZ and FASN inhibitor combination assays, AML and MM cell lines with varying sensitivities to CFZ were exposed to CFZ and FASN inhibitors, orlistat or TVB-3166, simultaneously, and the apoptosis rate were analyzed by flow cytometry. For western blots, selected AML and MM cell lines were incubated with compounds for 24 hours, and the lysates were probed for selected targets. Results: Single-agent CFZ induced apoptosis in sensitive AML and MM cell lines, while apoptotic rates remained low in resistant cell lines. Cell cycle analysis showed increased sub-G1 population in sensitive cell lines compared to resistant cell lines. RPPA revealed that FASN, a key enzyme involved in lipogenesis, correlated with CFZ sensitivity, and CFZ resistant lines trended towards higher basal FASN levels. When CFZ was combined with FASN inhibitors, orlistat or TVB-3166, significant synergy was observed in the apoptosis assays in the AML and MM cell lines. Western blot analyses showed FASN inhibitors enhanced the anti-proliferation and pro-apoptotic effects of CFZ. Conclusion: CFZ demonstrated single agent activity in nanomolar ranges in human AML and MM cell lines. When combined with agents targeting lipid-metabolism, CFZ showed synergistic effect in apoptosis, suggesting this combination could potentially be a new therapeutic strategy for AML and MM. Citation Format: Maoyu Peng, Sanaz Noelle Ghafouri, Martina SJ McDermott, Dennis J. Slamon, Sarah M. Larson. Fatty acid synthase (FASN) inhibitors synergize with carfilzomib (CFZ) in acute myeloid leukemia (AML) and multiple myeloma (MM) [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 3023.

Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhhibitor GX15-070 as a Novel Therapeutic Strategy in AML.

The Autocrine Secreted PDZD2 Functions As a Novel Tumor Suppressor in AML, Inducing Growth Inhibition and Cell Cycle Arrest