Vulnerabilities In Acute Myeloid Leukemia Research Articles

Anlotinib synergizes with venetoclax to induce mitotic catastrophe in acute myeloid leukemia

Venetoclax is a BCL2-targeted drug employed in treating various cancers, particularly hematologic malignancies. Venetoclax combination therapies are increasingly recognized as promising treatment strategies for acute myeloid leukemia (AML). In this study, we conducted an unbiased drug screen and identified anlotinib, a promising multi-targeted receptor tyrosine kinase inhibitor with oral activity currently utilized in the treatment of solid tumor, as a potent enhancer of venetoclax's anticancer activity in AML. Our investigation encompassed AML cell lines, primary cells, and mouse models, demonstrating effective low-dose combination therapy of anlotinib and venetoclax with minimal cytopenia or organ damage. Proteomic analysis revealed abnormal mitotic signals induced by this combination in AML cells. Mechanistically, anlotinib synergized with venetoclax by suppressing ARPP19 protein, leading to sustained activation of PP2A-B55δ. This inhibited AML cells from entering the mitotic phase, culminating in mitotic catastrophe and apoptosis. Additionally, we identified a specific synthetic lethal vulnerability in AML involving an ARPP19 mutation at S62 phosphorylation. These findings underscore the therapeutic potential of anlotinib and venetoclax combination therapy in AML, warranting further clinical investigation.

Translation efficiency driven by CNOT3 subunit of the CCR4-NOT complex promotes leukemogenesis

Protein synthesis is frequently deregulated during tumorigenesis. However, the precise contexts of selective translational control and the regulators of such mechanisms in cancer is poorly understood. Here, we uncovered CNOT3, a subunit of the CCR4-NOT complex, as an essential modulator of translation in myeloid leukemia. Elevated CNOT3 expression correlates with unfavorable outcomes in patients with acute myeloid leukemia (AML). CNOT3 depletion induces differentiation and apoptosis and delayed leukemogenesis. Transcriptomic and proteomic profiling uncovers c-MYC as a critical downstream target which is translationally regulated by CNOT3. Global analysis of mRNA features demonstrates that CNOT3 selectively influences expression of target genes in a codon usage dependent manner. Furthermore, CNOT3 associates with the protein network largely consisting of ribosomal proteins and translation elongation factors in leukemia cells. Overall, our work elicits the direct requirement for translation efficiency in tumorigenesis and propose targeting the post-transcriptional circuitry via CNOT3 as a therapeutic vulnerability in AML.

Tetraspanin CD81 Supports Cancer Stem Cell Function and Represents a Therapeutic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease associated with a poor outcome, necessitating aggressive therapies to achieve remission. A major contributing factor to treatment resistance and disease relapse is the presence of leukemic stem cells (LSC). Although challenging, targeting LSC constitutes an imperative approach to improve patient outcomes and limit side effects. In a previous study, we have uncovered a prognostic association between CD81 cell surface overexpression and worse outcome for AML patients. In the present work, we validated that CD81 overexpression of either protein or mRNA in AML blasts represents a specific prognostic marker for non-CBF AML with an extended cohort (n=150). We then confirmed our data using a publicly available dataset (BeatAML cohort). This association was also verified within NPM1 mutated patients, which was not associated to co- occurring FLT3-ITD mutations. When further investigating the underlying mechanisms related to CD81 overexpression and its impact on AML progression, we found out that CD81 expression was increased at relapse using paired diagnostic-relapsed samples (19% vs. 67%, P = 0.002). We demonstrated presence of CD81 within the LSC population, defined as CD34+/CD38-/CD90-/CD123+ blasts. Moreover, we showed that the CD81+ LSC fraction was higher at relapse compared to diagnosis (63% vs. 33%, P = 0.0001). In our cohort, non-CBF AML patients with higher level of CD81+LSC had worse outcome (RFS: P = 0.001, HR [95%CI] = 2.01 [1.16-3.48]; OS: P = 0.003; HR [95% CI] = 2.38 [1.32-4.29]. Next, we aimed to explore the potential impact of CD81 expression on LSC function, particularly focusing on its ability to serially engraft in immunocompromised mice (NSG). We observed a positive association between CD81 expression and an increased probability of positive engraftment, and a correlation with time-to-engraftment i.e., faster engraftment of xenografts from AML with high CD81 expression. Ex-vivo drug testing revealed primary AML cells as being more resistant to chemotherapy when engraftment was achieved. To investigate the mechanisms implied, we engineered either overexpression or knocked-down expression of CD81 in three different AML cell lines using cDNA transfection or shRNA, respectively. By means of these controlled CD81 expression models, we were able to demonstrate that CD81 overexpression increased AML cell adhesion, migration and invasion. Imaging by confocal microscopy exposed the development of membrane protrusions for CD81+ models. In vivo, higher CD81 expression increased blast homing, tumor burden and xenoengraftment efficiency. Moreover, we demonstrated that higher CD81 expression was linked to chemoresistance to anthracyclines and cytarabine, the two cornerstone drugs commonly used in AML. Finally, preclinical use of CD81 blocking antibody combined with chemotherapy in xenografts (AML cell line and patient-derived xenografts) resulted in longer relapse-free survival and reduced tumor aggressiveness. These results highlight the functional significance of CD81 overexpression in AML and its potential role as a biomarker for assessing LSC activity. Further investigations may improve this new avenue for targeted therapeutic intervention aimed at disrupting LSC function and improving treatment outcomes in AML patients.

COX4I1 Controls Mitochondrial Electron Transport Chain Complex IV Assembly and Leukemia Progression in Acute Myeloid Leukemia

Aberrant cellular signaling plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Identifying key signaling components and their dependencies is crucial for advancing AML therapy. Here, we conducted a focused CRISPR library screen targeting 427 cell signaling-related genes with 3,416 sgRNAs (8 sgRNAs per gene) in the Cas9-expressing Molm13 AML cells. In addition to the known leukemia essential genes (MYC, RPS6, CCND3, GAB2, and AURKB), we discovered COX4I1 (Cytochrome c oxidase subunit 4 isoform 1) as a novel essential gene for AML maintenance (Fig. 1). CRISPR-knockout of COX4I1 in AML cells led to inhibited cell proliferation, reduced colony formation, and attenuated leukemia progression in vivo. Transcriptomic profiling revealed elevated mitochondrial stress upon COX4I1 knockout, accompanied by dysregulated mitochondrial cristae structure observed through transmission electron microscopy. Functional assays demonstrated impaired mitochondrial respiration (oxygen consumption rate, i.e., OCR, measured by Seahorse XF) and depleted cellular ATP (measured by metabolomic mass spectrometry) upon COX4I1 knockout in AML cells, highlighting the pivotal role of COX4I1 in mitochondrial homeostasis and energy metabolism. COX4I1 is a nuclear-encoded mitochondrial protein located on the mitochondrial inner membrane. Using a high-density CRISPR gene tiling scan (Fig. 2; targeting density ~2.1 aa per sgRNA), we identified two critical COX4I1 peptide regions (P1: K67 - L73 and P2: E136 - K169) required for leukemia cell proliferation and mitochondrial respiration. Mechanistically, we found that expression of COX4I1 is essential for the assembly of mitochondrial complex IV, a crucial component of the electron transport chain involved in establishing the proton electrochemical potential across the mitochondrial inner membrane. Structural analysis revealed that facing the mitochondrial matrix, COX4I1's P1 region establishes the interface between COX4I1 and COX5A, which is necessary for the formation of the COX1 module. Conversely, COX4I1's P2 region (C-terminal 34 residues) is dispensable to the COX1 module formation; nonetheless, the P2 region co-localizes with the intermembrane space domains of the COX2/COX3 modules and is required for the assembling of an intact complex IV. Finally, we found that knockout of COX4I1 induced mitochondrial stress further triggers cellular ferroptosis in AML cells, which is accompanied by an imbalanced cellular redox index (reduced GSH/GSSG ratio) and elevated lipid peroxidation (increased malondialdehyde). These notions led us to discover the critical role of COX4I1 as an opportunity for targeted AML therapy, especially when combined with mitochondria-targeting therapeutics such as Venetoclax (i.e., ABT-199). In conclusion, our study highlights COX4I1 as a novel vulnerability in AML, controlling mitochondrial complex IV assembly and ATP biosynthesis. This finding holds promise for improving AML therapy and may be extended to identify therapeutically relevant signaling components in other hematopoietic malignancies.

Abstract 286: Targeting metabolic vulnerabilities in acute myeloid leukemia

Abstract Glutamine (Gln) is the most abundant amino acid intracellularly and in the plasma. Importantly, Gln depletion has emerged as a therapeutic approach for cancers that have undergone metabolic reprogramming and have exhibited a dependence on Gln for survival, including acute myeloid leukemia (AML). Asparaginases are bacterial enzymes that hydrolyze plasma glutamine to glutamate and ammonia, depriving glutamine-dependent AML cells from one of their vital nutrients. Preliminary data and literature have shown that following treatment with asparaginases in vitro there is a consistent increase in serine (Ser). Therefore, we hypothesized that in response to Gln depletion, AML cells upregulate de novo Ser biosynthesis as a compensatory pathway, and the inhibition of this pathway will synergize with Gln depletion to increase cytotoxicity in AML cells. We will test this by targeting, phosphoglycerate dehydrogenase (PHGDH), a key enzyme in Ser biosynthesis, which is a very common target for serine biosynthesis. We determined that crisantaspase (Rylaze) inhibited the proliferation of several AML cell lines with pharmacologically relevant IC50 values. Similarly, decreasing the concentration of Gln in the media resulted in a dose-dependent decrease in the proliferation, growth, and expansion of AML cells, confirming AML sensitivity to Gln depletion. Protein expression of PHGDH and PSAT was upregulated following Gln withdrawal from the media as well as following crisantaspase treatment. We set out to explore the effects of PHGDH knockout and observed that the PHGDH knockout AML cells were significantly more sensitive to crisantaspase, yielding IC50 values ranging from, ~250-fold lower compared to the parental lines. Gln depletion through crisantaspase treatment or withdrawal from the media decreased GSH levels and increased LDH release, both of which were further enhanced by PHGDH knockout. Indicating enhanced oxidative stress and cytotoxicity when both Gln and Ser are targeting in combination. Additionally, silencing of PHGDH reinforced anti-leukemic effects of glutamine modulation by different glutamine metabolism inhibitors, including V9302, CB-839 and BPTES. Preliminary testing of an NAD-dependent cell permeable PHGDH competitive inhibitor prodrug (BI4916) demonstrated an inhibition of AML cell proliferation in multiple cell lines. It has also demonstrated potential synergism and increase in cellular cytotoxicity when used in combination with Rylaze. Simultaneous targeting of Ser and Gln metabolism for AML treatment shows promising results in vitro with great potential for translation into clinical practice with clinically available asparaginases, and other glutamine modulators. Ongoing work is focused on the design of a more potent pharmacologic inhibitor of PHGDH to combine with crisantaspase as a novel treatment approach for AML. Citation Format: Kanwal Mahmood Hameed, Ashkan Emadi. Targeting metabolic vulnerabilities in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 286.

High MN1 Expression Is Associated with an Lspc-Enriched Phenotype and Glycolysis Representing a New Vulnerability in Acute Myeloid Leukemia

Despite a steady increase in the number of targeted therapies over the past decades, the 5-year overall survival (OS) rate remains poor in acute myeloid leukemia (AML). Currently, high expression of the oncogenic Meningioma 1 (MN1) is associated with poor OS in AMLs with normal karyotype, and we correlated its expression with a more immature cellular composition in AML samples. Hence, we hypothesized that supplementation of AML cells that usually fail to engraft in xenograft models, such as acute promyelocytic leukemia (APL), with MN1 would promote in vivo expansion in mice allowing the study of potential therapeutic vulnerabilities. The in vitro experiments were performed in human AML cell lines, primary AML cells (n=39), and isolated healthy CD34+ cells. MN1-overexpression (OE) was lentivirally performed. Bioenergetic profiles were evaluated by a Seahorse analyzer. Gene expression was measured by RNA-sequencing/qPCR. Protein levels were measured by immunofluorescence microscopy. Viability and surface protein expression was evaluated by flow cytometry. For in vivo studies, eight-weeks old female NSGS mice were transplanted with MN1-OE primary APL blast cells, and mice were followed for 12 weeks. Cells from the mouse organs were isolated, analyzed, and sorted for human APL blasts (CD45+CD117+CD33+) for ex vivo studies. We show that genetic MN1-OE in APL cells resulted in increased cell survival upon cytotoxic therapy, and decreased sensitivity to ATRA-induced terminal differentiation in short and long term cultures (3/8 days). In healthy CD34+ cells, we observed that MN1-OE was able to increase cell proliferation and stemness properties in CMP-sorted (CD34+CD38+CD123+CD45RA-) but not in GMP-sorted cells (CD34+CD38+CD123+CD45RA+), showing in human models the results from Heuser et al., 2011 in murine MN1-model. Co-expression of MN1+PML-RARα in healthy CD34+ cells resulted in superior expansion with retention of the CD34 marker, in comparison with the single hits. At the molecular level, APL cells with MN1high displayed transcriptional programs associated with "HYPOXIA"/"HSC-UP", whereas MN1low levels correlated with "OXPHOS-UP"/"GMP-UP". Despite no evident differences in Seahorse analysis, MN1-OE cells exhibited increased levels of the glycolytic enzyme PDK1. Functionally, MN1-OE cells were more sensitive to glycolytic inhibitors 2,2-dichloroacetophenone (a PDK1 inhibitor) and 2-deoxyglucose, revealing glycolysis as a metabolic vulnerability in MN1high AMLs, which was further validated in primary AML samples. These data are in line with our previous report indicating that more immature AML subtypes typically display a more glycolytic metabolic profile (Erdem et al, 2022). MN1 is an intrinsically disordered protein containing a long polyQ-stretch that is required for transformation (Riedel et al, 2021). We questioned whether the expression of chaperone proteins would be necessary for the appropriate functioning of MN1. We observed that expression of the DNAJB6 isoform 2 (DNAJB6b) correlated with MN1 expression across a large panel of genetic subtypes of AML, including more immature AML subtypes, such as the TP53-mutant AML but also in APL. shRNA-induced knockdown in AML cells of DNAJB6b resulted in cytosolic retention of MN1, associated with reduced cell survival. In vivo, the patient-derived xenograft model showed engraftment of APL samples upon MN1-OE, with the colonization of secondary organs and splenomegaly, which was not observed in the control. Flow cytometry analysis revealed that MN1-engrafted mice displayed increased levels of APL blasts in comparison with control, which had a large proportion of mature myeloid cells (CD33+CD11b+). Ex vivo screen of engrafted cells revealed increased resistance to ATRA-induced differentiation in MN1-OE sorted blasts. We provide further evidence to support the role of MN1 as an oncogenic hit allowing in vivo studies of difficult-to-engraft AML subtypes. Functionally, high MN1 expression resulted in decreased differentiation, retention of stem-like properties, and metabolic shift towards a more glycolytic state. We demonstrated that DNAJB6b controls the cellular localization of MN1, with its downregulation associated with the loss of MN1 supportive functions. Finally, our findings suggest that targeting glycolysis can be leveraged as a therapeutically actionable mechanism for driving cell death in MN1high AMLs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dietary methionine starvation impairs acute myeloid leukemia progression

AbstractTargeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, nuclear magnetic resonance−based metabolite quantifications and 13C-tracing, polysomal profiling, and chromatin immunoprecipitation sequencing, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis, and induced a cell cycle block. Reactive oxygen species levels were not increased following methionine depletion, and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.

Unleashing Cell-Intrinsic Inflammation as a Strategy to Kill AML Blasts.

This study exploits inflammatory programs inherent to AML blasts to identify genetic vulnerabilities in this disease. In doing so, we determined that AML cells are dependent on the transcriptional repressive activity of IRF2BP2 for their survival, revealing cell-intrinsic inflammation as a mechanism priming leukemic blasts for regulated cell death. See related commentary by Puissant and Medyouf, p. 1617. This article is highlighted in the In This Issue feature, p. 1599.

Mannose Metabolism Is a Metabolic Vulnerability Unveiled By Standard and Novel Therapies in Acute Myeloid Leukemia

IntroductionThe development of resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML). Metabolic rewiring has emerged as a therapeutically actionable vulnerability in AML, where specific metabolic adaptations arising as a result of driver mutations or in response to therapy have been reported. Mannose phosphoisomerase (MPI) is the first enzyme in the mannose metabolism (MM) pathway, that leads to the production of GDP-Mannose, a key sugar donor for N-glycosylation reactions. MPI was amongst the top drop-out genes in our published CRISPR-Cas9 screen aiming to identify sensitizers to FLT3 tyrosine kinase inhibitors (TKI) in AML carrying activating FLT3 internal tandem duplication (ITD) mutations (Gallipoli et al., Blood 2018). Moreover, analysis of published genomic datasets indicates that MPI expression levels are higher in AML compared to normal samples, correlate with patient outcome (A) and further increase in paired relapsed to diagnosis samples. We therefore hypothesized that MM and MPI inhibition sensitize AML cells to both FLT3-TKI and standard chemotherapy and tested this in preclinical models.MethodsExperiments were performed in human AML cell lines, primary AML mononuclear cells and normal CD34 + stem/progenitor cells. Liquid chromatography coupled to mass spectrometry and oxygen consumption/extracellular acidification rate as measured by a Seahorse analyser were employed to assess metabolic changes. Gene-expression was measured by RNA-sequencing and confirmed by RT-qPCR. Viability, surface protein expression and lipid peroxidation were assessed by flow-cytometry. Protein expression and localisation was measured by western blot and immunofluorescence. Gene silencing was performed using CRISPR-Cas9 gene editing and inducible short hairpin RNA interference.ResultsWe show that genetic and chemical MPI inhibition sensitizes FLT3 WT and FLT3 ITD AML cell lines in vitro to standard cytarabine chemotherapy and FLT3-TKI respectively and these effects are rescued by the addition of MPI downstream product mannose. We validate these findings in vivo using MPI knock-out (KO) cell line xenografts and in vitro using primary AML samples following MPI knock-down (B). We also demonstrate a lack of toxicity to normal CD34 + cells.Surprisingly, global metabolomic analysis show that MPI KO cells accumulate fatty acids and particularly polyunsaturated fatty acids (PUFA) (C), due to both increased uptake and reduced fatty acid oxidation (FAO). MPI KO cells downregulate several FAO metabolism genes and this is corroborated by the strong positive correlation of MPI expression levels with multiple genes involved in FAO across multiple primary AML datasets. Metabolic profiling demonstrates that MPI KO cells have reduced oxidative phosphorylation metabolism and in particular are unable to oxidise palmitate, an effect rescued by mannose or the FAO activator fenofibrate (D).We link this metabolic defect to the specific activation of the ATF6 arm of the unfolded protein response (UPR) due to a reduction in protein glycosylation in MPI KO cells. We validate this by demonstrating that activating or inhibiting ATF6 respectively phenocopies or rescues the effects of MPI KO (E) and show this to be secondary to ATF6 driving FAO inhibition, via transcriptional downregulation of PPARα, a master regulator of lipid catabolism.Finally, we show that accumulation of PUFA in MPI KO cells is accompanied by reduced cysteine levels and increased markers of oxidative stress and lipid peroxidation, such as 4-hydroxynonenal (4-HNE), all features associated with ferroptosis (F). We validate this by showing that combining FLT3-TKI with MPI KO leads to ferroptotic cell death in AML cells, which can be rescued by the radical-trapping antioxidant ferrostatin.ConclusionsWe provide further evidence to support the role of metabolic rewiring in driving therapy resistance in AML and show for the first time that targeting MPI and MM sensitizes AML cells to cytarabine and FLT3-TKI. Mechanistically we unveil a novel connection between MM and fatty acid metabolism, via activation of the ATF6 arm of the UPR, leading to cellular PUFA accumulation, lipid peroxidation and ferroptotic cell death. Finally, our findings also suggest that triggering ferroptosis can be leveraged as a therapeutically actionable mechanism driving cell death in therapy-resistant AML cells. [Display omitted] DisclosuresVassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.

An In Vivo CRISPR Screening Platform to Identify New Therapeutic Targets in AML

First-generation, large-scale functional genomic screens have revealed hundreds of potential genetic vulnerabilities in acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because these large-scale genetic screens were primarily performed in vitro in established AML cell lines, their translational relevance has been debated. Therefore, we established a protocol for CRISPR screening in orthotopic xenograft models of human AML, including patient-derived-xenograft (PDX) models that are tractable for CRISPR-editing.We first defined experimental conditions necessary for an optimal in vivo screen via barcoding experiments. We determined that sub-lethal irradiation was necessary for improved barcode representation in bone marrow and to reduce mouse-to-mouse variation. Moreover, it was critical to combine samples from multiple mice to achieve complete in vivo library representation. Next, using the Broad DepMap and other publicly available functional genomic screen data, we identified 200 genes that were stronger dependencies in AML cell lines compared to all other cancer types screened. Using this list, we created a secondary library and performed parallel in vivo and in vitro screens using the MV4-11 and U937 cell lines and a PDX model. In vitro and in vivo hits were surprisingly well correlated, although a modest number of targets did not score well in vivo. Notably, dependencies identified across AML cell line models were strongly recapitulated in the PDX model, validating the application of AML cell lines for dependency discovery.Our in vivo screens nominated the mitochondria-localized RING-type ubiquitin E3 ligase MARCH5 as a potential therapeutic target in AML. Using CRISPR, we first validated this in vitro dependency on MARCH5 and determined that MARCH5 is a critical guardian to prevent apoptosis in AML. MARCH5 depletion activates the canonical mitochondrial apoptosis pathway in a BAX/BAK1-dependent manner. Multiple genome-wide screens revealed that a dependency on MARCH5 is strongly correlated with a dependency on MCL1, but not other anti-apoptotic BCL2-family members, across the AML cell lines in the screen. As observed with MCL1 inhibition, MARCH5 depletion sensitized AML cells to venetoclax, a BCL2-specific inhibitor FDA-approved in combination with a hypomethylating agent for the treatment of older adults with AML. Importantly, MARCH5 depletion diminished the venetoclax resistance induced by MCL1 overexpression but not that caused by BCLXL overexpression. Altogether, these results suggest that MARCH5 is required for maintaining MCL1 activity specifically.Since there are no small molecule inhibitors directed against MARCH5, we deployed a dTAG system as an approximation of pharmacological inhibition. This approach uses a hetero-bifunctional small molecule that binds the FKBP12 F36V-fused MARCH5 and the E3 ligase VHL, leading to the ubiquitination and proteasome-mediated degradation of the fusion protein. dTAG-MARCH5 cells were established via deleting endogenous MARCH5 by CRISPR and expressing exogenous FKBP-tagged MARCH5 protein. MARCH5 degradation with the dTAG molecule dTAG V-1 markedly impaired cell growth in vitro. Additionally, we demonstrated the utility of dTAG system in vivo using a PDX model. The combination treatment of dTAG V-1 and venetoclax elicited a much stronger anti-leukemic effect compared to the treatment with only venetoclax or dTAG V-1, further highlighting MARCH5 as a promising synergistic target for enhancing the efficacy of venetoclax in AML.Taken together, our in vivo screening approach, coupled with CRISPR-competent PDX models and dTAG-directed protein degradation, constitute a useful platform for prioritizing AML targets emerging from in vitro screens to serve as the starting point for therapy development. DisclosuresDharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: Bristol Myers Squibb: Consultancy; KronosBio: Consultancy; AstraZeneca: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.

Myeloid Leukemia Dependencies at CTCF-Enriched Long Noncoding RNA Loci

The noncoding genome presents a largely untapped source of biological insights, including tens of thousands of long noncoding RNA (lncRNA) loci. While some produce bona fide lncRNAs, others exert transcript-independent cis-regulatory effects, and the lack of predictive features renders their mechanistic dissection highly challenging. Here, we describe CTCF-enriched lncRNA loci (C-LNC) as a putative new subclass of functional genetic elements exemplified by MYNRL15 - myeloid leukemia noncoding regulatory locus on chromosome 15.Initially identified by an expression-guided CRISPRi screen of hematopoietic stem and progenitor (HSPC) / acute myeloid leukemia (AML) lncRNA signatures (480 genes, 1545 sgRNAs), we found MYNRL15 dependency in myeloid leukemia cells of diverse genetic backgrounds. Interestingly, cis and trans perturbation approaches revealed both the MYNRL15 transcript and its flanking protein-coding genes to be dispensable. High density CRISPR tiling of a 15 kb area centered on MYNRL15 (1613 sgRNAs) instead uncovered two crucial, candidate cis-regulatory DNA elements in the locus, which drive the MYNRL15 perturbation phenotype. To determine the molecular basis of MYNRL15 dependence, we performed transcriptome, chromatin conformation, chromatin accessibility, and CTCF profiling. RNA-sequencing established MYNRL15's involvement in maintaining key cancer dependency pathways (e.g. cell cycle, ribosome, spliceosome). Further, MYNRL15 perturbation associated with the coordinated dysregulation of several chromosome 15 neighbourhoods, and formation of a long-range chromatin interaction between the locus and the base of a distal loop, as detected via next-generation Capture-C. The gained interaction was accompanied by diffuse gains in chromatin accessibility across the distal interaction sites (ATAC-seq) as well as reduced CTCF occupancy at the MYRNL15 locus (CTCF CUT&RUN), altogether indicating the 3D re-organization of chromosome 15 following MYNRL15 perturbation. Integrative analysis of the chromatin conformation and transcriptome data, combined with a small CRISPR-Cas9 knockout screen of protein-coding genes from the gained interaction region (29 genes, 149 sgRNAs), pinpointed two potent cancer dependency genes that are located in the region and downregulated following MYNRL15 perturbation: namely, WDR61 and IMP3. Individual knockout of both genes robustly depleted myeloid leukemia cells, recapitulating the MYNRL15 perturbation phenotype and positioning WDR61 and IMP3 as its regulatory targets. Importantly, in primary cells, MYNRL15 perturbation eradicated AML blasts while sparing 50-60% of CD34 + HSPCs in vitro, and reduced patient-derived AML xenografts up to 10-fold in vivo, indicating a potential therapeutic window.Having implicated MYNRL15 in 3D genome organization and demonstrated its role in myeloid leukemia cells, we explored whether MYNRL15 may belong to a sub-category of biologically relevant lncRNA loci that have thus far been overlooked due to their lack of transcript-specific functions. Remarkably, elevated CTCF density (e.g. number of CTCF binding sites per kb of gene length) distinguishes MYNRL15 and 531 other lncRNA loci in K562 cells, of which 43-54% associate with genetic subgroups and/or survival in AML patient cohorts, and 18.4% are functionally required for leukemia maintenance as determined by CRISPR-Cas9 screening. The latter hit identification rate represents a substantial improvement over typical lncRNA essentiality screens (which range from 2-6%) - illustrating the effectiveness of CTCF density metrics in refining functional lncRNA candidate lists, and underlining the relevance such loci hold for AML and cancer pathophysiology in general. Curated C-LNC catalogs in other cell types will facilitate the search for noncoding oncogenic vulnerabilities in AML and other malignancies. [Display omitted] DisclosuresReinhardt: Celgene Corporation: Consultancy; Novartis: Consultancy; Bluebird Bio: Consultancy; Janssen: Consultancy; CLS Behring: Research Funding; Roche: Research Funding. Klusmann: Bluebird Bio: Consultancy; Novartis: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy.

Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition.A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis.To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells.Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. DisclosuresMinden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

A KDM4A-PAF1-mediated epigenomic network is essential for acute myeloid leukemia cell self-renewal and survival

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

SIRT5 IS A DRUGGABLE METABOLIC VULNERABILITY IN ACUTE MYELOID LEUKEMIA.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.

TFEB links MYC signaling to epigenetic control of myeloid differentiation and acute myeloid leukemia.

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML) and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs, by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by a MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.

A Synthetic Lethal Approach to Eradicate AML Via Synergistic Activation of Pro-Apoptotic p53 By MDM2 and BET Inhibitors

Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually compared to many other cancers, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation via MDM2i and minimize toxicity, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. These screens identified BRD4 inhibition as a candidate synthetic lethal partner of MDM2i. BRD4 is a member of the Bromodomains and Extraterminal (BET) family of proteins, a transcriptional co-activator and already a candidate AML therapeutic target. Surprisingly, we found inhibition of BRD4 alone induces expression of some of p53 target genes. We unexpectedly reveal that BRD4 binds to p53 target genes and acts as a transcriptional repressor of these genes. Synergistic cell killing by the drug combination (MDM2i + BET inhibitor (BETi)) depends on synergistic activation of p53 target genes, such as PUMA and NOXA, due to simultaneous stabilization of p53 by MDM2i and relief of BRD4-mediated repression by BETi. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type TP53in vitro, against two mouse models of AML in vivo, and against primary human patient blasts in vitro. Furthermore, we used BET PROTACs to selectively and completely induce degradation of BRD4 in cells. Consistent with results from BETi, BET degraders and MDM2i synergize to suppress cell viability with superior potency. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, co-targeting wild-type TP53 and a transcriptional repressor function of BRD4 represent a novel synthetic lethal vulnerability in AML. Disclosures Latif: AbbVie: Consultancy, Honoraria; Takeda UK: Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Kite: Consultancy, Honoraria, Speakers Bureau; Jazz: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria. Higgins:Roche: Current Employment, Current equity holder in publicly-traded company, Other: Support of parent study and funding of editorial support. Copland:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Roche: Research Funding; Epizyme: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; Gilead: Speakers Bureau.

Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers

AbstractMetabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non–caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.

Therapeutic Vulnerabilities in Acute Myeloid Leukemia (AML) with TP53 Alterations

Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow with a low two‐year survival rate. Standard chemotherapy achieves complete remission in 60–80% of patients, but 20–30% patients relapse due to leukemia stem cells (LSCs) that can self‐renew and recapitulate disease. AML with TP53 alterations has a lower prognosis with survival rates of 0–10% at one year. In vitro drug screens have identified drugs to target AML, but none have studied the effect of these therapies on LSCs with TP53 alterations. These in vitro drug screens include Crizotinib, Elesclomol, AZD1480, GW2580, Venetoclax, and Entrectinib. Crizotinib, AZD1480, GW2580, Venetoclax, and Entrectinib are signaling pathway inhibitors. Elesclomol induces oxidative stress and apoptosis in cancer cells. The goal of this research is to understand the molecular mechanisms of self‐renewal and therapeutic vulnerabilities in LSCs of AML with TP53 alterations. Specifically, we investigate whether these agents target LSCs from AML with TP53 alterations. We performed in vitro viability and in vitro colony forming assays (CFAs) on primary human AML samples with TP53 alterations, plating them with drugs or vehicle control. Furthermore, we used CYTOF to assess the effects of each drug on signaling within leukemia subpopulations. Crizotinib proved to be effective in reducing in vitro viability, in vitro CFAs, and affecting the expression of immunophenotypic markers, such as CD34 and CD123, which are markers for leukemia stem cell populations. Our data reveals cell‐type specific effects of Crizotinib in human AML with TP53 alterations. Our findings suggest that Crizotinib may be an effective therapy for patients with AML with TP53 alterations.Support or Funding InformationThis research was supported and funded by: NIH/NIGMS MARC U* STAR T34 HHS 00026 National Research Service Award to UMBC, Summer Research at the University of Minnesota Medical School NIH HLBI 2R25HL088728‐11A1, Dr. Colin Campbell, Dr. Craig Henke, American Cancer Society Mentored Research Scholar Grant (MSRG‐16‐195‐01‐DDC), Frederick A. Deluca Foundation, CTSI K to R01 Award, NIH/NCATS, Lois and Richard King Assistant Professorship in Medicine, Division of Hematology, Oncology, Transplantation, Dept. of Medicine, University of Minnesota University of Minnesota Foundation Donors

Loss of Kat2a enhances transcriptional noise and depletes acute myeloid leukemia stem-like cells.

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy with abnormal progenitor self-renewal and defective white blood cell differentiation. Its pathogenesis comprises subversion of transcriptional regulation, through mutation and by hijacking normal chromatin regulation. Kat2a is a histone acetyltransferase central to promoter activity, that we recently associated with stability of pluripotency networks, and identified as a genetic vulnerability in AML. Through combined chromatin profiling and single-cell transcriptomics of a conditional knockout mouse, we demonstrate that Kat2a contributes to leukemia propagation through preservation of leukemia stem-like cells. Kat2a loss impacts transcription factor binding and reduces transcriptional burst frequency in a subset of gene promoters, generating enhanced variability of transcript levels. Destabilization of target programs shifts leukemia cell fate out of self-renewal into differentiation. We propose that control of transcriptional variability is central to leukemia stem-like cell propagation, and establish a paradigm exploitable in different tumors and distinct stages of cancer evolution.

Integrating Enhancer Profiling and CRISPR Dropout Screen Revealed Selenoprotein Synthesis Pathway As a New Vulnerability in AML

Alterations of the epigenetic landscape and transcription are hallmarks of acute myeloid leukemia (AML) that drive leukemogenic gene expression and therefore can be exploited for therapeutic intervention. To look for such targets that harbor both an altered epigenetic feature and are genetically essential for AML cells, we performed a multi-database analysis integrating pan-cancer super enhancer landscapes with whole genome CRISPR dropout screens. Among the top targets, we discovered SEPHS2. An enhancer was present upstream of the gene marked by H3K27ac and bound by leukemogenic transcription factors including MYB, Pu.1 and RUNX1. In addition, AML cells with SEPHS2 deletion significantly dropped out in a genome wide CRISPR screen. This gene encodes a critical enzyme in the underappreciated selenoprotein synthesis pathway which was highly upregulated in TCGA AML patients compared to control blood cells from healthy individuals. Collectively, our initial bioinformatic analysis suggested that the selenoprotein synthesis pathway is a new vulnerability in AML. To test the functional requirement of the selenoprotein synthesis pathway in AML and other cells, we performed CRISPR mediated deletion of three key genes in the selenoprotein synthesis pathway, SEPHS2, SEPSECS and EEFSEC. The human AML cell lines (MOLM13, THP1 and Kasumi1), murine AML cells transformed by MLL-AF9 and human AML PDX cells all depicted a significant dependency on these genes, while proliferation of normal cord blood cells and myeloma cells (U266B1) was almost not affected. We then transplanted these cells into recipients. Deletion of SEPHS2, SEPSECS or EEFSEC significantly ameliorated AML progression, indicated by decreased AML burden and extended survival. Since selenoproteins including GPX1 and GPX4 are known antioxidants, we hypothesized that perturbing the selenoprotein synthesis pathway disrupted the redox state in AML cells. We found that deletion of SEPHS2, SEPSECS or EEFSEC elevated ROS in AML cells as demonstrated by Cell-Rox or DCF-DA staining. Western blotting revealed significantly downregulated GPX4 level and upregulated DNA damage marker γ-H2AX. Moreover, the defective proliferation was partially rescued by adding antioxidant TEMPOL. These results suggested selenoprotein synthesis pathway produced key antioxidants to balance the proper redox state and was required for AML cell proliferation. A major source of selenium is diet. Therefore, we hypothesized that consuming selenium low diet could suppress AML. We compared the survival of AML bearing mice on selenium proficient and deficient diet. The selenium deficient diet significantly extended survival, lowered GPX4 level and increased ROS in AML cells. Interestingly, normal mouse on selenium deficient diet for a 3-months period did not develop any abnormalities in CBC or bone marrow hematopoiesis. This suggests selenium deficient diet could be clinically applicable without significant side effects. Altogether, the integration of a pan-cancer enhancer landscape study with CRISPR dropout gene screen offered a powerful tool to dissect cancer targets that possessed unique enhancer features and genetic essentiality. The analysis yielded SEPHS2 and its selenoprotein synthesis pathway to be a new vulnerability in AML. The underappreciated selenoprotein synthesis pathway was key to produce antioxidant selenoproteins such as GPX1 and GPX4 to maintain a proper redox state in AML. Deleting the genes or removing selenium from diet could perturb the pathway and ameliorate the AML disease. Disclosures Lin: Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.