OXPHOS Inhibition via LUC7L2 as a Target for SF3B1-Mutant Myelodysplastic Syndrome

Background: SF3B1 gene mutations are the most common spliceosome mutations seen in myelodysplastic syndrome (MDS) patients. Though it is well known SF3B1 mutations cause downstream changes in erythroid differentiation and the cell cycle, which leads to malignancy, metabolic changes arising from this mutation are unknown. Our preliminary data shows that patient samples harboring an SF3B1 mutation exhibit alternative splicing of the gene LUC7L2. Interestingly, this gene encodes for another protein of the spliceosome complex: U1 snRNP. The gene LUC7L2 has been shown to regulate metabolic processes by promoting glycolysis while repressing oxidative phosphorylation (OXPHOS) via various downstream mechanisms. We therefore hypothesize that SF3B1-mutant MDS could result in alternative splicing of the gene LUC7L2, thereby altering the metabolic dependencies of these cells towards OXPHOS. Methods: RNA sequencing was performed to compare four SF3B1-mutant MDS patient samples with non-mutant ones. LUC7L2 was found to be alternatively spliced and downregulated in SF3B1 mutant cells. Genetic inhibition of LUC7L2 was then performed in the non-mutant MOLM-13 myeloid malignant cell line using siRNA technology, and a Western blot was performed to ensure siRNA inhibition was successful. Seahorse assays were then performed to assess oxidative phosphorylation upon knockdown of LUC7L2. Results: OXPHOS is increased in MOLM-13 myeloid malignant cells when LUC7L2 is inhibited. The results suggested that this gene, which is alternatively spliced and shows lower expression in SF3B1-mutant MDS, increases myeloid malignant dependence on OXPHOS. To test whether SF3B1-mutant cells are dependent on OXPHOS for survival, they were treated with oligomycin, an ATP synthase inhibitor. Mutated cells show less viability than the wild type suggesting an increased dependence on OXPHOS. Conclusion: With lower expression of LUC7L2 in SF3B1-mutant MDS cells, OXPHOS becomes the main source of energy production in these cells, and it is therefore a potential therapeutic target

OXPHOS Inhibition via LUC7L2 as a Target for SF3B1-Mutant Myelodysplastic Syndrome

SF3B1 gene mutations are the most common spliceosome mutations seen in myelodysplastic syndrome (MDS) patients. Though it is well known SF3B1 mutations cause downstream changes in erythroid differentiation and the cell cycle, which leads to malignancy, metabolic changes arising from this mutation are unknown. We conducted RNA sequencing from SF3B1-mutant MDS patient samples and found several genes related to metabolism were alternatively spliced. Of these, LUC7L2 was selected as our target as previous studies show its involvement in promoting oxidative phosphorylation (OXPHOS) via various downstream mechanisms when knocked down.We show that OXPHOS is increased in MOLM-13 myeloid malignant cells when LUC7L2 is inhibited. The results suggested that this gene, which is alternatively spliced and shows lower expression in SF3B1-mutant MDS, increases myeloid malignant dependence on OXPHOS

Myogenic carbohydrate transcriptome in murine C2C12 cells

Murine C2C12 cells, derived from adult dystrophic mouse thigh muscle simulate in vivo myogenesis (Yaffe and Saxel, 1977). They undergo three distinct stages of C2C12 myogenesis: proliferative myoblasts (day 0); cell cycle withdrawal and fusion to become early myotubes (day 4); and, finally spontaneously contracting late myotubes (day 9). The present study analyzed the changes in the transcriptome of the enzymes involved in carbohydrate metabolism during the three different stages of myogenic development of C2C12 cells. A carbohydrate metabolism qRT-PCR array was used to examine the mRNA levels of 84 different enzymes involved in carbohydrate metabolism (n=4 per stage). A total of 64 genes showed changes in mRNA levels that were statistically significant (ANOVA, p\u3c0.05) from the control group (day 0). Of these 64 genes, 29 genes increased or decreased by two (2.0) fold or higher and were considered to be biologically significant changes. Eleven genes were involved in the TCA cycle, 5 genes were involved in glycolysis, 7 genes were involved in glycogen metabolism, 4 genes were involved in the pentose phosphate pathway and two genes were involved in gluconeogenesis. Isoenzyme mRNA level shifts were also present during the three stages of myogenesis. In addition, the enzymatic activities of citrate synthase (CS) and creatine kinase (CK) were measured (n=6) during each stage. CS activity was 112, 204, and 271 nmol/min/mg total protein and 37, 62 and 105 nmol/min/mg total DNA respective to day 0, 4, and 9. CK activity was 86, 342, and 532 nmol/min/mg total protein and 29, 105, 661 nmol/min/mg total DNA respective to day 0, 4, and 9. These data indicate that the TCA cycle, glycolysis and glycogen metabolism pathways were significantly altered during myogenesis to meet the energetic and synthetic needs of developing cells

4353 The Role of BCL2 Mediated Calcium Signaling on Leukemia Stem Cell Metabolism

OBJECTIVES/GOALS: The objective of this study is to define the molecular mechanisms that control survival of malignant stem cells in acute myeloid leukemia (AML). Leukemia stem cells (LSCs) are not effectively eradicated by standard treatment and lead to resistance and relapse, which contribute to poor survival rates. METHODS/STUDY POPULATION: The recently FDA approved venetoclax, a BCL2 inhibitor, with azacitidine, a hypomethylating agent leads to a 70% response rate in AML patients. Analysis of patients treated with this regimen showed direct targeting of LSCs. BCL2 has a non-canonical function in regulation of intracellular calcium. To determine how BCL2 mediated calcium signaling plays a role in LSC biology, we used LSCs isolated from venetoclax/azacitidine (ven/aza) sensitive and resistant patient samples to measure expression of calcium channels via RNA seq. BIO-ID, siRNA, flow cytometry, seahorse assays, calcium measurements and colony assays were used to determine the effects of calcium channel perturbation on LSC biology. RESULTS/ANTICIPATED RESULTS: BCL2 inhibition leads to decreased OXPHOS activity in primary AML specimens. BIO-ID studies revealed cation/metal ion transporters, ER membrane proteins and ER membrane organization as top enriched pathways interacting with BCL2. RNA-seq data showed increased expression of genes involved in calcium influx into the ER in ven/aza sensitive LSCs and increased expression of genes involved in calcium efflux from the ER in ven/aza resistant samples. Ven/Aza resistant LSCs have increased mitochondrial calcium content, consistent with their increased OXPHOS activity as calcium is required for OXPHOS. Perturbation of these channels leads to decreased OXPHOS activity and decreased viability in LSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: We postulate that a deeper understanding of the mechanisms behind ven/aza targeting of LSCs will lead to the development of novel therapies for patients who do not respond to ven/aza. Our data show targeting intracellular calcium signaling could be a viable therapeutic strategy for AML patients.</jats:p

STAT3 Plays a Critical Role in Mitochondrial Function and Survival of Primary AML Cells

Introduction: Acute myeloid leukemia (AML) is an aggressive disease with a dismal prognosis. This is largely due to high relapse rates, which stem from our inability to eliminate leukemia stem cells (LSCs) with conventional chemotherapy. As we have gained a deeper understanding of the biology of LSCs, new targets against these chemo-resistant cells have surfaced. One such example is treatment using venetoclax/azacititine, a therapy that has significantly improved the outcome for these patients. Notwithstanding, some patients show resistance to all treatments, and developing a larger repertoire of agents that target LSCs remains an unmet need in this disease. One key vulnerability of LSCs is their dependence on oxidative phosphorylation (OXPHOS). Although signal transducer and activator of transcription 3 (STAT3) has been classically studied as a transcription factor that regulates self-renewal and proliferation, it has also been shown to play an essential role in OXPHOS via regulation of the electron transport chain (ETC). Given that STAT3 is commonly overexpressed in AML, and LSCs are dependent on OXPHOS, we hypothesized that STAT3 may be an effective target for eradication of LSCs. Methods: We have developed a novel small molecule inhibitor of STAT3, SF25. This compound, as well as genetic knockdown of STAT3, was employed to test the functional role of STAT3 in primary AML specimens. Flow cytometry, colony-forming potential, and engraftment of primary samples in PDX mouse models were performed to assess therapeutic efficacy. RNA-seq, seahorse assays, and metabolomics experiments were also performed to determine molecular mechanisms linked to targeting STAT3. Results: Our data shows that inhibition of STAT3 in primary AML samples leads to decreased cell viability, colony-formation and engraftment potential in xenograft models, while not affecting normal hematopoietic stem cells. This effect appears to be a result of mitochondrial dysfunction in LSCs, as seen by a significant decrease in oxygen consumption rate of STAT3 depleted cells. The mitochondrial dysfunction and reduction in OXPHOS is mediated by the downregulation of several mitochondrial and nuclear encoded genes that are important for oxidative phosphorylation, including several electron transport chain complex genes. Inhibition of STAT3 also affects glutaminolysis, as noted by metabolomics analysis of leukemia stem cells treated with STAT3 inhibitor. We suspect this effect is mediated by down-regulation of Myc upon STAT3 inhibition, which blocks glutamine conversion to glutamate, and leads to further decrease in TCA cycle intermediates. Conclusions: Acute myeloid leukemia is an aggressive disease, largely due to the presence of a chemo-resistant population of leukemia stem cells. LSCs highly depend on proper mitochondrial function and OXPHOS, a process that is partly regulated by STAT3 via multiple mechanisms. We propose that inhibition of STAT3 is therefore an effective way of eliminating this population, making this a promising new target in the treatment of AML. Disclosures No relevant conflicts of interest to declare. </jats:sec

KRAS insertion mutations are oncogenic and exhibit distinct functional properties.

Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-Ras(G60_A66dup) and K-Ras(E62_A66dup) proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications

Lysosomal Acid Lipase a (LIPA) Modulates Leukemia Stem Cell (LSC) Response to Venetoclax/TKI Combination Therapy in Blast Phase Chronic Myeloid Leukemia

Abstract Chronic myeloid leukemia (CML) is a heterogeneous disease, initiated by reciprocal translocation of chromosome 9 and 22, resulting in the generation of a BCR-ABL fusion protein and constitutive activation of the ABL kinase. ABL tyrosine kinase inhibitors (TKIs) have been very successful in suppressing CML disease. However, TKIs may not eliminate leukemia stem cells (LSCs), as evidenced by the frequent re-emergence of the disease upon TKI discontinuation. Moreover, blast phase CML (bpCML) remains a formidable challenge in disease management. Recent clinical evidence suggests that the BCL2 inhibitor venetoclax (Ven) in combination with ABL-targeting tyrosine kinase inhibitors (TKI) can eradicate bpCML LSCs. However, the exact mechanism by which this combination may targets LSCs is not known. In this report, we confirm the efficacy and LSC-targeting capacity of Ven/TKI combination in preclinical models of bpCML and we further identify that inhibition of free fatty acid (FFA) mobilization pathways may provide enhanced efficacy against LSCs. To establish the efficacy of Ven/TKI combination, we treated bpCML samples with Ven+Dasatinib (Das) combination for 24h, this resulted in a significant decrease in the viability of bulk and primitive populations (CD34+, CD38+). Patient-derived xenografts of bpCML samples in NSGS-mice, were treated with Ven/Das as well as single agents. The result showed a significant decrease in leukemia burden in the combination treated group, compared to either drug alone, albeit, some resistant cells survived in the combo treated group. Furthermore, using a syngeneic mouse model of bpCML, co-expressing Bcr-Abl and Nup98-HOXA9 translocations, the mouse leukemic cells treated with Ven/Dasatinib combination demonstrated a significant loss of viability of the bulk as well as phenotypically defined LSCs (Lin-/Sca1+). Treatment of leukemic mice with Ven/Das had a significant survival benefit and remained disease free at 80 days post treatment. We also showed significant survival benefits of Ven/ponatinib in NSGS-mice harboring syngeneic bpCML cells with the T315I gatekeeper mutation. Treatment of normal mice with Ven/Das combo did not affect the colony forming ability of LSK cells from the bone marrow, indicating a leukemia-specific response. Based on these results, we conclude that Ven/TKI combination effects were due to direct targeting of the LSC population. To investigate the potential mechanism of Ven/TKI activity in LSC targeting, we performed gene expression studies using RNA-seq based methods after short term treatment. Our findings indicated that the LSC population from Ven/TKI-treated mice showed enrichment of a gene signature associated with lysosome biology. Pre-treatment of mouse leukemia cells with bafilomycin, an inhibitor of lysosome function, resulted in increased sensitivity to Ven/TKI combo. Intriguingly, we also found significant induction of lysosomal acid lipase (LIPA), an enzyme involved in the generation of free fatty acids for energy needs. Metabolomic analysis of LSCs isolated after short term treatment with Ven/TKI, showed that a number of fatty acids were up-regulated in the Ven/Das treated group compared to control. Knocking down Lipa using CRISPR technology resulted in increased sensitivity to Ven/TKI combination, whereas overexpression of Lipa resulted in decreased sensitivity to the Ven/TKI combination, implicating Lipa upregulation and a resultant increase in free fatty acids as a protective response to Ven/TKI treatment. Furthermore, knocking down CPT1A, an important free fatty acid mitochondrial transporter, resulted in increased sensitivity to Ven/TKI combination both in mouse and primary human leukemic cells, leading to the hypothesis that activation of fatty acid processing through enhanced Lipa activity may represent a compensatory response to venetoclax based therapies in bpCML. In summary, we demonstrate the preclinical efficacy of Ven/TKI combination therapies for targeting of bpCML LSCs. Furthermore, our data suggest that blocking upregulation of free fatty acids through mechanisms such as inhibition of LIPA activity, might synergize with Ven/TKI combinations to eradicate LSCs, allowing for more durable response. Our findings provide a therapeutic rationale for blocking pathways involved in free fatty acids generation, as a potential strategy for increasing remission duration. Disclosures Pollyea: Amgen: Consultancy; Janssen: Consultancy; Genentech: Consultancy; AbbVie: Consultancy, Research Funding; Syndax: Consultancy; Daiichi Sankyo: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Celgene/BMS: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Glycomimetics: Other. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding. </jats:sec

The STAT3-MYC axis promotes survival of leukemia stem cells by regulating SLC1A5 and oxidative phosphorylation

Abstract Acute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.</jats:p