Grant support for the development of peasant farms: the experience of the Trans-Baikal Territory and key problems

The article considers the current state of small and medium-sized businesses in rural areas of the Trans-Baikal Territory. In the region, the largest share of agricultural products produced falls on the personal subsidiary farms, while there is a destruction of large-scale commodity production, evidenced by a decrease in the number of agricultural enterprises and organizations. Presently, the Government of the Trans-Baikal Territory is interested in transforming personal subsidiary farms into the status of peasant farms, since they are able to ensure more efficient functioning of small forms of farms. Special attention is paid to the state support of small farms in the region. In the course of the study, the problems of the effectiveness of the use of state support for small forms of management were identified. The priority direction of state support is grant subsidization of peasant farms, while the distribution of grants in the context of municipal districts of the region is uneven. In general, the mechanism of grant support for peasant farms is effective only for areas of the region where animal husbandry traditionally constitutes the basis of agriculture. There are also problems associated with excessive requirements for obtaining grants and insufficient consulting assistance from potential grantees.

YOUNG WOMEN FARMERS IN THE SCOPE OF THE YOUNG FARMERS GRANT SUPPORT PROJECT: THE CASE OF KIRIKKALE PROVINCE IN TURKIYE

This study aimed to reveal the situation of young women farmers (YWFs) who benefited from cattle farming support for three years in Kırıkkale, one of the provinces where the study was carried out. In Kırıkkale province, 397 young farmers were supported and 250 of them were YWFs. The projects with the highest grant support were cattle farming projects, and they constituted 62.22% of the projects (247 units). The rate of YWF who benefited from cattle farming support for three years was higher than young men farmers (YMF) and was determined as 59.51%. In this study, face-to-face survey questionnaires were filled in the 2020 year with 36 YWFs and 36 YMFs. As a result of the study, it was determined that YMFs have more experience in cattle breeding than YWFs. It is seen that especially YWFs are married and their families have high non-agricultural income; their husbands support especially YWFs at the application stage. 52.78% of YWFs and 69.44% of YMFs stated that they want to expand their farms with the given support. As a result of the study, it was determined that there was a significant increase in the number of animals after the given support to the young farmers, and it was revealed that the most important problem of the young farmers was that they had financial difficulties in the supply of production inputs. It is seen that this project, which has both social and economic aspects, encourages YWFs to take more part in agricultural activities. However, it is important to determine more effective criteria at the selection stage, follow up and supervise the beneficiaries of the incentives both during and after the project, and support the successful ones to grow their farms. Keywords: Young women farmers, young farmers project support, rural development, kırıkkale-Turkiye.

Grant support as an option for private sector solutions and systemic issues in the work of NPOs

This article examines the existing practice of participation by non-profit organizations (NPOs) in solving social policy problems, both in the West and in Russia, and makes a comparative analysis of Russian and foreign experience in this area. A separate section of the work is devoted to the study of the grant support system of NPIs in Russia. Systemic and private problems have been identified. Conclusions are drawn about imperfections of the emerging system of interaction between the State and NCBs, including in the area of project financing; and also about the need to create conditions for the replication of successful pilot projects of individual NCBs on the territory of the whole of Russia.

FLT3 Inhibitors Upregulate CXCR4 and E-Selectin Ligands and CD44 Via ERK Suppression in AML Cells, and Blockade of CXCR4 and E-Selectin Signaling with GMI-1359 Overcomes AML Resistance to Quizartinib in Vitro and In Vivo

FLT3 inhibitors (FLT3i) have had transient success treating FLT3-mutant acute myeloid leukemia (AML) patients, especially those with FLT3 internal tandem duplication (ITD) mutations that account for one-third of adult AML cases (Daver et al., 2021). However, FLT3i are typically ineffective in eliminating leukemia stem cells in the protective bone marrow (BM) microenvironmental "niche" (Borthakur et al., 2011; Cortes et al., 2013; Zhang et al., 2008). Cytokines and chemokines such as CXCR4 and E-selectin ligands play a critical role in leukemia cell protection in the BM niche. Indeed, interactions of leukemic cells with their vicinal support cells, including mesenchymal stem cells (MSCs) and endothelial cells (ECs), in the BM niche is mediated mainly through the CXCR4/SDF-1 and E-selectin/HECA-452/CD44 axes (Erbani et al., 2020; Peled and Tavor, 2013). Therefore, evaluation of the effects of FLT3i on CXCR4 and E-selectin signaling in leukemia cells could enhance our understanding of AML FLT3i resistance mechanisms. To this end, we investigated the levels of CXCR4 and E-selectin ligands on FLT3-ITD-mutated AML cells in vitro and in vivo during FLT3i treatment (e.g., quizartinib or sorafenib), and evaluated the anti-leukemia effects of CXCR4/E-selectin blockade with the dual inhibitor GMI-1359. We first checked the effects of FLT3i on the levels of CXCR4 and E-selectin ligands, as well as CD44, in vitro in human MOLM14 AML cells, which harbor FLT3-ITD mutations. All of these were upregulated, as measured by flow cytometry, following exposure to quizartinib (p < 0.001) or sorafenib (p < 0.01) for 96 h. The mRNA levels were also increased roughly 2-fold, as measured by qPCR, suggesting transcriptional regulation was involved in the upregulation. Further, the upregulation of CXCR4 and E-selectin ligands and CD44 was time dependent (from 2 to 96 h). FLT3i profoundly suppresses activation of ERK, AKT, and Stat5 (Zhang et al., 2008). Therefore, we tested if the suppression of each signaling pathways individually could upregulate of CXCR4. Unexpectedly, 72-h suppression of MEK/ERK signaling with selumetinib or pimasertib also upregulated CXCR4 in MOLM14 cells. No effects in this regard were observed by suppressing AKT/mTOR or Stat5 with AZD8055 or STAT5-IN-1, respectively. Additionally, in Dox-inducible NRAS (G12D)-mutated MOLM13 AML cells which also harbor FLT3 ITD mutations, ERK activation by doxycycline downregulated CXCR4 levels implying the MEK/ERK signaling pathway was associated with the suppression of CXCR4. Furthermore, under BM microenvironment-mimicking, co-culture using human MSCs/ECs and MOLM14 cells, blockade of CXCR4 and/or E-selectin signaling using the CXCR4 antagonist plerixafor, the E-selectin antagonist GMI-1271, or the CXCR4 and E-selectin dual inhibitor GMI-1359 showed that GMI-1359 markedly abrogated BM protection and sensitized MOLM14 cells to quizartinib-induced apoptosis. We further validated the effect of GMI-1359 in a PDX model of AML which were from a patient who relapsed from sorafenib+E6201+DAC in clinic and showed resistant to quizartinib ex vivo. The combination of GMI-1359 with quizartinib profoundly reduced leukemia burden and extended survival of the PDX mice compared to the vehicle or the single-agent treatments (median survival was 158 days vs. 82.5, 79 and 128 days, respectively, in combination group vs. vehicle, quizartinib and GMI-1359; p < 0.0001) [Figure 1,2]. Our results suggest that FLT3i can upregulate CXCR4 and E-selectin ligands and CD44 in FLT3-ITD leukemia cells, which is mediated, at least in part, via suppression of MEK/ERK signaling. GMI-1359 sensitized AML cells to quizartinib-induced apoptosis in vitro and statistically significantly extended AML PDX mouse survival in vivo. These findings provide a pre-clinical rational for using GMI-1359 to prevent or overcome FLT3i resistance when treating FLT3-mutant AML patients. Figure 1 Figure 1. Disclosures Fogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Konopleva: Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Forty Seven: Other: grant support, Research Funding; KisoJi: Research Funding; AstraZeneca: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Ablynx: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Calithera: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Stemline Therapeutics: Research Funding; Ascentage: Other: grant support, Research Funding. Andreeff: Senti-Bio: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; AstraZeneca: Research Funding; Karyopharm: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; ONO Pharmaceuticals: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Medicxi: Consultancy; Oxford Biomedica UK: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy.

Targeting Polo-like Kinase 4 Triggers Polyploidy and Apoptotic Cell Death in TP53-Mutant Acute Myeloid Leukemia

Background: TP53 mutations in acute myeloid leukemia (AML) are associated with complex karyotype, high incidence of minimal residual disease (MRD), and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018). While numerous novel treatment regimens, including the combination of the BCL2 inhibitor venetoclax (VEN) and hypomethylating agents (HMA), have emerged as partially effective treatments and resulted in higher remission rates in patients with TP53-mutant AML, full clearance of the mutant TP53 clone is rarely achieved and the majority of patients relapse (Short et al., 2021; Takahashi et al., 2016). The mechanisms responsible for response and relapse in TP53-mutant AML remain unclear and investigating novel mechanisms is critical to develop more effective therapies. Results: In order to shed light on the defective p53 signaling pathways underlying TP53 mutant AML, and to better understand mechanisms of resistance, we performed RNA-sequencing (RNA-seq) on FACS-sorted subpopulations using samples collected from TP53-mutant or TP53-wt high-risk AML patients. Samples were collected at diagnosis (DX) and post-treatment (POSTTX) (total number of samples n= 67, TP53-mutant=36, TP53-wt=31). Diagnostic samples include bulk AML, leukemic stem cells (LSCs), and post-treatment samples including bulk mononuclear cells (MNCS) and patient specific MRD (total n= 67, DX_Bulk=15, DX_LSCs=15, POSTTX_MNCs=14, POSTTX_MRD=23). Differential gene expression analysis of TP53-mutant samples indicates a positive enrichment of the following pathways: G2/M checkpoint, MYC targets, and mitotic spindle, among others. We focused here on genes associated with TP53-mutant AML enriched pathways, and identified a key regulator of centriole biogenesis, one of E2F targets: Polo-like kinase 4 (PLK4) as a potential target highly expressed in TP53-mutant AML samples . Previous publications showed that PLK4 is transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing (Fischer et al., 2014; Li et al., 2005). Here we show that TP53-mutant AML samples lack the p53-dependent PLK4 repression and have higher levels of PLK4 compared to TP53-wt AML. To test the rigor of this finding, we interrogated the Munich Leukemia Laboratory (MLL) data base and analyzed their clinically annotated (e.g. karyotype, survival, complete blood counts, previous treatments ... etc) RNA-seq dataset of 726 AML samples (TP53-mutant=72, TP53-wt=654). TP53-mutant AML samples consistently showed significant PLK4 upregulation (p= 0.0003). We analyzed PLK4 expression and its correlation with TP53 mutations in The Cancer Dependency Map project dataset (1375 cell lines in 35 different types of cancers) (p= 0.004). Furthermore, we found significantly higher PLK4 protein levels in TP53-mutant AML MOLM13 cell lines when compared with syngeneic TP53-wt AML MOLM13 cells. Experimentally, we found that PLK4 inhibition using 25nM CFI-400945 in TP53-mutant AML MOLM13 cell lines triggers polyploidy > 2-fold higher than in TP53-wt AML MOLM13 cell lines 72 hours post treatment (Fig.1A p< 0.0001). Finally, we show that polyploidy is not reversible after drug removal and results in significantly increased levels of apoptotic cell death in TP53-mutant AML MOLM13 cells (Fig.1B). Conclusion: Our data suggest that TP53-mutant AML expresses higher levels of PLK4 in comparison to TP53-wt AML, and targeting PLK4 triggers polyploidy and apoptotic cell death in TP53-mutant AML. A clinical trial is ongoing testing the efficacy of PLK4 inhibition (CFI-400945) in AML (Clinical Trial ID: NCT04730258, TWT-202). Figure 1 Figure 1. Disclosures Issa: Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Novartis: Consultancy, Research Funding. Borthakur: Takeda: Membership on an entity's Board of Directors or advisory committees; ArgenX: Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; Astex: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Konopleva: Ascentage: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Stemline Therapeutics: Research Funding; KisoJi: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Sanofi: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Andreeff: Senti-Bio: Consultancy; ONO Pharmaceuticals: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy; Breast Cancer Research Foundation: Research Funding; Oxford Biomedica UK: Research Funding; Karyopharm: Research Funding; Medicxi: Consultancy; Amgen: Research Funding; AstraZeneca: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Syndax: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

Enhanced p53 Activation By Dual Inhibition of MDM2 and XPO1 Disrupts MYC Transcriptional Program and Restores Sensitivity to BCL-2 Inhibition in Ven/HMA Resistant AML

The BCL-2 inhibitor venetoclax (ven) in combination with hypomethylating agents (HMA) has revolutionized acute myeloid leukemia (AML) therapy. However, the majority of patients with AML who received ven/HMA eventually relapse and cures are still elusive. We previously generated ven-resistant MV4;11 (MV4;11 VR) cells and found them to express elevated c-Myc protein levels. The dual inhibition of MDM2 and XPO1 significantly increased nuclear p53 protein and dramatically upregulated p53 target genes. On the other hand, dual inhibition of MDM2 and XPO1 profoundly decreased c-Myc protein levels in a p53-dependent manner, resulting in the profound downregulation of MYC transcriptional program. Clinical grade MDM2 and XPO1 inhibitors milademetan (mil) and selinexor (sel) significantly reduced leukemia burden and prolonged survival in the xenograft model injected with MV4;11 VR cells. However, the treatment effect did not result in very long relapse free responses. To investigate potential resistance mechanisms to dual inhibition of MDM2 and XPO1, we utilized our previously reported (Muftuoglu, ASH 2020) multiparameter flow cytometry to assess multiple stress responses pathways including p21, ATF4, PARP, LC3B, Ki-67, active caspase-3 and amine-reactive viability dye at the single cell level in ven-resistant AML cells. We found that residual cells, after combined MDM2 and XPO1 inhibition, expressed high levels of p21, ATF4 and LC3B and low Ki-67 levels, suggesting that the resistant population is in a cell kinetic quiescent state with activation of autophagy and ER stress. Interestingly, ven-resistant MV4;11 cells with in vivo acquired resistance to dual inhibition of MDM2 and XPO1 demonstrated elevated protein levels of respiratory chain complex proteins (NDUFB8, MTCO1, UQCRC2 and ATP5A), suggesting increased OXPHOS dependency. Intriguingly, these MV4;11VR cells with subsequently acquired resistance to MDM2 and XPO1 inhibition in vivo have restored sensitivity to ven. We previously reported that the combination of MDM2 and BCL-2 inhibitors induces synergistic apoptosis through the elimination of dormant p21 high residual AML cell (Pan, Cancer Cell 2017). In a clinical trial of Idasanutlin + Venetoclax we observed over 45% response rates in relapsed/refractory AML patients (Daver, ASH 2020). Therefore, we hypothesized that combining MDM2/XPO1 inhibition with BCL-2 inhibition further induces cell killing in ven/HMA resistant AML cells. To address this, we treated ven-resistant AML cells with triple combination of mil, sel and ven, resulting in a profound cytoreduction in vitro compared with other single and doublet treatments. Next, we treated NSG mice injected with PDX AML cells with FLT3-ITD, GATA2 and NRAS mutations obtained from a patient who relapsed after ven/decitabine treatment. The doublet combinations especially of mil + ven reduced circulating blasts and significantly prolonged survival. Of note, no mice have died at day 180 after treatment in the group receiving triple combination of mil, ven and sel, with profound and sustained cytoreduction (more than 2 log 10 difference) and significantly prolonged survival compared with any other treatment (Fig. A, B). The triple combination was well-tolerated and did not decrease mouse CD45+ cells, platelets and hemoglobin. In conclusion, the concomitant inhibition of MDM2, XPO1 and BCL-2 was feasible and exerted dramatic and sustained anti-leukemia activity in ven/HMA resistant AML cell in vitro and in vivo. Milademetan (RAIN-32) is currently being developed by Rain Therapeutics. Figure 1 Figure 1. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Daver: Pfizer: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Novartis: Consultancy; Gilead Sciences, Inc.: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; Sevier: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Novimmune: Research Funding; Hanmi: Research Funding; Amgen: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Lesegretain: Daiichi-Sankyo Inc.: Current Employment. Seki: Daiichi-Sankyo Inc.: Current Employment. Shacham: Karyopharm: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: (8999996, 9079865, 9714226, PCT/US12/048319, and I574957) on hydrazide containing nuclear transport modulators and uses, and pending patents PCT/US12/048319, 499/2012, PI20102724, and 2012000928) . Konopleva: Ascentage: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; AstraZeneca: Other: grant support, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; KisoJi: Research Funding; Ablynx: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Sanofi: Other: grant support, Research Funding. Andreeff: Medicxi: Consultancy; Glycomimetics: Consultancy; AstraZeneca: Research Funding; Syndax: Consultancy; Amgen: Research Funding; Oxford Biomedica UK: Research Funding; Karyopharm: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Senti-Bio: Consultancy; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; ONO Pharmaceuticals: Research Funding; Aptose: Consultancy.

Accumulation of Intracellular L-Lactate and Irreversible Disruption of Mitochondrial Membrane Potential upon Dual Inhibition of Oxphos and Lactate Transporter MCT-1 Induce Synthetic Lethality in T-ALL Via Mitochondrial Exhaustion

Metabolic reprogramming is recognized as one of the key hallmarks in acquiring aggressive phenotype and chemoresistance in solid tumors and hematologic malignancies. We have previously demonstrated that T-ALL are characterized by significant dependency on oxidative phosphorylation (OxPhos) with ability to utilize glutamine either in oxidative or reductive directions of TCA cycle, when mitochondria are blocked by Complex I Inhibitor (Baran N, et al. ASH 2020). To survive upon Complex I blockade leukemic cells require functional monocarboxylate transporter MCT1, that enables excretion of lactate and permissive pyruvate flux (Fig.1 a). Here we show that metabolic intervention utilizing OxPhos blockade can be potentiated by targeting MCT1 transporter and propose a novel metabolic synthetic lethality that could be exploited to eradicate T-ALL and other OxPhos-dependent malignancies. We first demonstrated that Complex I inhibition leads to increased MCT1 expression; on the contrary, MCT1 transporter blockade forces cells to increase OxPhos. In turn, the combinatorial therapy with Complex I inhibitor (IACS-010759) and MCT1 inhibitor (AZD3965) causes loss of ATP content (Fig. 1b), significant reduction of cell number and massive induction of apoptosis. Mechanistically, the combination treatment further reduced oxygen consumption rate (OCR) (Fig. 1c) and increased extracellular acidification rate, as measured by Seahorse. In concert with those results, dual inhibition led to TCA blockade, accumulation of intracellular lactate and depletion of glutamine, cystathionine and glutathione, indicating severe disruption of redox balance as measured by mass spectrometry and confirmed by significant accumulation of intracellular and mitochondrial reactive oxygen species (ROS) (Fig. 1d), loss of mitochondrial membrane potential (ΔΨ) (Fig. 1e) and subsequent mitochondria swelling. RNAseq data showed simultaneous upregulation of glycolysis and glutathione-related processes as possible mechanisms of metabolic compensation, yet strong upregulation of genes regulating apoptosis related to mitochondria dysfunction (Fig. 1f). Real-time hyperpolarized MRI based metabolic imaging studies with [1-13C]-pyruvate in patient-derived xenografts in vivo revealed significant decrease of lactate-to-pyruvate ratio in mice treated with AZD3965 or IACS-010759 alone, and in mice treated with drug combination. [13C]-Glucose isotope tracing analysis in patient-derived xenografts in vivo revealed an increased intracellular trapping of lactate as a marker of treatment effectiveness in mice subjected to dual blockade. While MCT1 inhibition induced only moderate reduction of leukemia growth in vitro and tumor burden in vivo, combination with IACS-010759 depleted significantly both, circulating and marrow/spleen/liver resident leukemia cells. Mechanistically, inhibition of MCT1 by AZD3965 therapy in leukemia-bearing mice led to lactate accumulation, OCR increase, moderate ROS production and mitochondrial membrane hyperpolarization, while Complex I blockade resulted in upregulation of MCT-1, reduction of OCR, lactate production and increase of ROS ; consequently, combinatorial therapy caused complete mitochondria shut-down and drastic inhibition of tumor growth both in vitro and in vivo in two xenografts models and led to significant extension of overall survival (p<0.0001) (Fig. 1g). In summary, these results demonstrate a novel synthetic vulnerability of concomitant blockade of OxPhos and MCT-1, uncovering metabolic checkpoints that can ultimately translate into successful therapies in T-ALL and OxPhos-dependent malignancies. Figure 1 Figure 1. Disclosures Skwarska: Halilovich E, Wang Y, Morris E, Konopleva M, Skwarska A.: Patents & Royalties: Combination of a MCL-1 inhibitor and midostaurin, uses and pharmaceutical composition thereof.. Konopleva: Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Ascentage: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Cellectis: Other: grant support; Sanofi: Other: grant support, Research Funding; KisoJi: Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support.

Cigarette Smoke or Cigarette Condensate Exposure Accelerates Growth of FLT3-ITD AML Models, Induces Oxidative Stress, and Alters DNA Methylation

Acute myeloid leukemia (AML) patients with any history of cigarette smoking have worse survival outcomes compared to patients that have never smoked. The molecular basis of cigarette smoking or cigarette smoke exposure (CSE) impacting AML progression or treatment response is unknown. Altered DNA methylation from smoking persists decades after quitting and has been followed in peripheral blood mononuclear cells (PBMCs) but have not been evaluated in the context of AML. Smoking also causes oxidative stress in PBMCs, but this has yet to be studied in AML patients with a history of smoking. To model how smoking worsens AML progression, we created a CSE model using a cigarette smoking robot where NOD-SCID mice received 2 hours of CSE 5 days/week or air alone. After 2 weeks of CSE, 100,000 luciferase-tagged, human AML cells were injected via tail-vein and leukemic burden was monitored by bioluminescent imaging. Two cell lines, MOLM13 and MOLM14 carrying the oncogenic fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation, when introduced into CSE mice had enhanced leukemic progression within one week (p-value <0.0001 and <0.001 respectively). MOLM14-bearing mice showed increased leukemic burden 24 days post injection (p-value<0.05). DNA methylation was evaluated by bisulfite sequencing of splenocytes from CSE mice, which was mapped to the human (AML) or mouse (host) genome. Over 200 significant changes in DNA methylation of gene promoters was seen, including hypomethylation of aryl-hydrocarbon receptor repressor (AHRR), an established hallmark of smoking in humans. Indicating that our CSE model recapitulates DNA methylation from smoking in humans. Additionally, GATA-2, a critical protein for hematopoietic differentiation known to be frequently mutated in AML, was also amongst the top hypomethylated genes. We quarried TCGA to understand the implications of altered DNA methylation in AML patients. In AML patients, low GATA-2 methylation showed decreased survival compared to those with high GATA-2 methylation (N=42/group, p-value: 0.000138). The discovery of GATA-2 methylation in smoking models and its attribution as a poor prognostic indicator in AML is novel, which underscores a need to understand the role of GATA-2 methylation in AML. Reactive oxygen species (ROS) have been implicated in AML progression, drug resistance, and are elevated in otherwise healthy smokers. No significant differences were seen in intracellular ROS in spleen or PBMCs of CSE mice; however, we found more than a two-fold increase of heme oxygenase 1 (HO-1) (p-value:0.0505), a protein involved in antioxidant responses and AML treatment resistance. There was also increased BCL-2 protein expression and a decrease in AHRR expression (p-value: 0.0098) in CSE mouse samples. This suggests that CSE causes oxidative stress and increases pro-leukemic signaling. To address if CSE impacted treatment efficacy, we treated mice with daunorubicin (2 mg/kg, twice weekly via tail-vein) once evidence of engraftment was detected. We found a trend towards increased leukemic burden compared to non-smoking mice which approached statistical significance. To study the direct impact of CSE on AML, without exposure to the tumor environment, AML cells were treated in vitro with a commercially available cigarette smoke condensate (CSC) that contains the chemicals from cigarette smoke. To mimic the in vivo CSE, MOLM13 cells received two weeks of CSC treatment before being injected into mice. At 3, 10, and 17 days post injection, mice with CSC-treated AML had enhanced leukemic burden (p-value <0.0001, <0.0001, and <0.001). This model revealed that chemicals in cigarette smoke can directly promote AML. On day 14 of CSC treatment, mirroring when the cells were injected into mice, both FLT3-ITD cell lines had increased ROS levels and or glutathione as measured by flow cytometry; this is indicative of CSC-induced oxidative stress. Cumulatively, these data define novel changes in DNA methylation and redox from smoking or tobacco products with strong potential to drive AML progression and therapy resistance. Future studies will determine if blocking these redox or methylation events can reverse the accelerated AML growth and will aid in the creation of a tailored treatment strategy for AML patients with any history of smoking. Disclosures Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Konopleva: Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; AstraZeneca: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; KisoJi: Research Funding; Stemline Therapeutics: Research Funding; Agios: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Cellectis: Other: grant support; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding. DiNardo: Novartis: Honoraria; Agios/Servier: Consultancy, Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; Takeda: Honoraria; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding.

Diverse Mechanisms of Resistance to Decitabine and Venetoclax Therapy in Newly Diagnosed and Relapsed/Refractory AML Inferred By Transcriptome Analysis

A selective BCL-2 inhibitor venetoclax synergizes with hypomethylating agents (HMA). Combination of venetoclax plus HMAs (decitabine or azacitidine) is FDA approved for the treatment of high-risk newly-diagnosed (ND) AML including older patients and patients unfit intensive chemotherapy. We have reported that combination of venetoclax with ten-day HMA decitabine (DEC10-VEN, NCT03404193) demonstrated high efficacy in ND AML patients, with lower overall response rates in relapsed/refractory (R/R) AML. Specifically, the complete remission (CR/CRi) rates were 84% in ND AML and 42% in R/R AML (DiNardo et al. Lancet Haematol. 2020). The determinants of reduced efficacy in the R/R compared to ND AML are not defined. To characterize the transcriptomic signatures associated with response or resistance to DEC10-VEN in ND vs R/R AML, we performed RNA-seq analysis on samples from genetically diverse 24 AML patients treated on DEC10-VEN clinical trial. The pre-treatment samples from 11 ND patients, including 6 patients who achieved CR/CRi and 5 who failed to respond or subsequently relapsed (non-responders, NR); and from 9 R/R patients, 3 patients who achieved CR/CRi and 6 NR, have been analyzed with a NovaSeq 6000 platform. We first performed the comparative analysis of NR vs CR/CRi cases using pre-treatment samples. In ND AML patients 113 genes were higher and 62 genes were lower, and in R/R group 33 genes were higher and 69 genes were lower, in non-responders than in CR/CRi patients (log fold-change >|2|, FDR < 0.05). In ND AML, the top functional processes identified by Gene Ontology (GO) analysis (FDR < 0.05) in non-responders were genes involved in oxidative phosphorylation (OXPHOS) and cell migration/adhesion, such as upregulation of OXPHOS/energy metabolism-associated genes MT-CO3, COX6B2, NADK and cell-to-cell interactions mediator TSHBS1. In contrast, R/R AML patients that failed to achieve the response had lower transcriptomic signatures of genes associated with immune system such as TLR8, FCER2, TNFAIP3, CCR7, compared to responders. Recently, HMAs have been reported to activate silenced endogenous retroviral (ERV) elements regulated by epigenetic modifications, followed by upregulation of interferon-response genes and activation of energy metabolism through ATP hydrolysis (Fresquet et al. Cancer Discovery, 2021). ERVs re-activation may have dual opposing role in malignant cells including leukemia: (1) inducing "immunogenic cell death" by mounting innate antiviral immune responses; and (2) promoting carcinogenesis through activation of Notch signaling and fostering tumor cell proliferation with energy consumption. We analyzed transcription of 45 well-annotated ERV in 9 samples of 6 pre- and 3 post- DEC10-VEN treatment in R/R AML patients who failed to respond. The differential expression analysis with the custom-built ERV reference genome by STAR demonstrated ERVFRD, HERVK-27 and ERVK3-1 as significantly upregulated after DEC10-VEN treatment. Of note, HERV-K27 is one of human cancer-associated HERV-K proviruses. GO analysis demonstrated that these 3 ERVs are positively associated with innate immune system, Notch, Wnt, TGF beta signaling pathways and fatty acid oxidation. To further determine the potential function of ERVs, we investigated the upregulated genes after DEC10-VEN treatment in R/R AML that failed to respond. The top 10 significantly upregulated GO terms were dominated by immune response and cytoplasmic translation, associated with upregulation of ATP hydrolysis activating gene TNNT1, interferon simulated gene TNFAIP3, Notch1 regulating CAMK2A, TGF beta regulating SMAD7, and endogenous RNA MUEOV that promotes tumor metastasis through TGF beta signaling. Taken together, our transcriptomic analysis suggests that diverse pathways may contribute to the development of resistance to DEC10-VEN in AML cells in ND and R/R patients. In ND patients, high OXPHOS and microenvironment confer resistance to therapy. In R/R AML, baseline immune suppression is aided by the ERVs reactivation by DEC10-VEN therapy, which in turn may promote ATP consumption, energy metabolism, and Notch signaling (Figure 1). These findings are currently being validated and may identify novel mechanisms of resistance to DEC10-VEN treatment in ND and R/R AML. Figure 1 Figure 1. Disclosures DiNardo: Foghorn: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; Forma: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria. Konopleva: Ablynx: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; AstraZeneca: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Cellectis: Other: grant support; KisoJi: Research Funding; Calithera: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Forty Seven: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights.