Abstract Background: Multiple studies have confirmed the central role of preexisting immune response measured by stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC). Emerging studies showed that not only the number of TILs but also the location of TILs is important. There are 3 distinct immune architectures described based on the amount and locations of TILs, namely immune enriched (IN), immune excluded (IE), and immune desert (ID). Here we evaluated outcomes and characteristics associated with each immune landscape. Methods: NanoString Digital Spatial Profiling (DSP) and CosMx, a spatial multi-omics single-cell imaging platform, were performed in 75 samples from the Mayo Clinic (MC) TNBC cohort (Leon-Ferre BCRT 2018). NanoString IO360 was performed in 114 samples from the FinXX trial ( NCT00114816) . Firstly, tumors with sTIL quantified by H&E ≤ 30% were classified as ID. The rest of the tumors with high sTIL > 30% were categorized according to the intratumoral CD8 protein expression by DSP, with IE having intratumoral CD8 in the lower median and IN having intratumoral CD8 in the upper median. Chi-square test, gene set enrichment, Cox regression, and Kaplan-Meier analysis were used. Differential expression listed as log 2-fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p< 0.05. Results: ID is associated with low Ki67 < 5% (23.3% vs. 9.6% ID, p 0.02) as well as apocrine (11/13, 84.6%) and metaplastic histology (10/12, 83%). In both univariate and multivariate analysis, patients with IN had significantly improved recurrence-free survival (RFS) compared to those with ID (HR 0.37, 95%CI 0.18-0.74, p 0.005). Despite having high sTILs, IE had poor outcomes similar to ID (HR 0.84, 95%CI 0.36-1.98). Strikingly, we identified that IE patients had significantly lower plasmacytoid dendritic cells (pDCs) compared to IN (mean 0 vs. 0.26/100 tumor cells, 95%CI 0.08-0.43 , p 0.01). Using Gene Set Enrichment Analysis to evaluate differential hallmarks between IN and IE, we identified IF Nɑ and IFNγ (FDR < 0.001) responses as significantly enriched in IN group, consistent wi th the function of pDCs, which are a subset of dendritic cells specialized in secreting high levels of type I interferon. To validate this finding, we further evaluated the 11 leading edge gene IFNɑ signature in the FinXX trial. A high IFNɑ signature score was associated with significantly improved outcomes in the FinXX trial (HR 0.21, 95%CI 0.09-0.51 , p < 0.001). Similar findings were observed using Kaplan-Meier analysis in the FinXX trial with significantly improved RFS (p 0.0006) and overall survival (p 0.0001) in patients with high IFNɑ signature scores. Furthermore, we evaluated the differential gene expression unique to IN tumors in the Mayo cohort. Expressions of MHC class I and class II in tumor cells, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DPA1, and HLA-E, were associated with IN and significantly improved outcomes (p < 0.05) compared to ID and IE. Conclusions: Highlighting the importance of spatial context, we identified that patients with IE tumors had poor outcomes despite having high TILs. Moreover, u sing an in-depth analysis with spatially defined context, we identified the central role of pDC and the significance of IF Nɑ in TNBC. Support: Breast Cancer Research Foundation, Mayo Clinic Breast Cancer SPORE (P50CA116201-17) W81XWH-15-1-0292, P50CA015083, R35CA253187 Citation Format: Saranya Chumsri, Yi Liu, Yaohua Ma, Jodi Carter, Mark Gregory, Sarah Church, Jason Reeves, Heather Ann Brauer, Sarah Warren, Heikki Joensuu, Edith Perez, Roberto Leon-Ferre, David Hillman, Judy Boughey, James Ingle, Krishna Kalari, Fergus Couch, Matthew Goetz, Keith Knutson, E. Thompson. The spatially resolved single-cell atlas of the tumor immune architecture revealed the central role of IFN-alpha and plasmacytoid dendritic cells in triple-negative breast cancer in the Mayo Clinic cohort and FinXX trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-03.

Abstract Background: Tumor progression and therapeutic response are regulated by the tumor microenvironment. Understanding the spatial association and architecture of molecular characteristics and composition of tumor immune microenvironment at single-cell and subcellular resolution encourages improvements in clinical prognosis and immunotherapy benefits. Single-cell Spatial profiling technologies permit the study of transcriptional activity at the spatial, single-cell level and provide abundant, high-resolution information required for the identification of clinical-related features in immuno-oncology. Methods: We performed an ultra-high-plex RNA assay to detect 6,000 targets simultaneously in situ on FFPE human skin squamous cell carcinoma using the CosMx™ Spatial Molecular Imager (SMI). For the selection of regions of interest, four protein markers and DAPI were co-detected on the same tissue slide. Tertiary analysis algorithms were developed for cell typing, co-localization of genes and ligands, cell-cell interaction, and pathway analysis. Results: Thousands of transcripts were simultaneously detected with high sensitivity and specificity on the FFPE skin squamous cell carcinoma tissue section at single-cell subcellular resolution. We investigated the tumor microenvironment including cell types, their spatial distribution and proportion of diversified immune cells in the tumor compartment. The cell typing results revealed many cancerous subpopulations, which resolved spatially from cells that lacked any defining marker genes. We also assessed distances between immune cells and their nearest functional-related neighbors. Moreover, we revealed the ligands co-localization, as well as spatial patterns of direct cell-cell interactions and signaling pathways. We found that some genes have elevated expression in macrophages which are near cancer compared to those are far from cancer cells, empowering the molecular investigation of novel signaling between immune and cancer cells in tumor immune microenvironment. Conclusions: Single-cell spatial measurements of 6,000 RNA and four proteins on the same tissue section, along with a large viewing area on archival FFPE tissue, provide a novel tool to reveal the spatial signature of tumor microenvironment and oncogenic pathways, facilitating the next level of cancer and therapeutic research. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Michael Patrick, Shanshan He, Saskia Ilcisin, David Kroeppler, Patrick Danaher, Jason Reeves, Mark Gregory, Haiyan Zhai, Michael Rhodes, Joseph Beechem. Uncover spatial signatures of tumor microenvironment and oncogenic pathways using 6,000-plex single-cell spatial molecular imaging on FFPE skin squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3644.

Although FLT3 inhibitors (FLT3i) improve median survival of patients with Acute Myeloid Leukemia (AML), they will eventually acquire resistance and relapse. Clinical trials have revealed that AML blasts in the bone marrow (BM) were not effectively eliminated compared to AML blasts in peripheral blood, suggesting a protective role of the BM microenvironment that counteracts FLT3i therapy. Our group discovered that BM stromal cells protect FLT3-mutated AML cells from the killing effect FLT3i through upregulation of Ataxia Telangiectasia Mutated (ATM), which promotes Mammalian Target of Rapamycin Complex 1 (mTORC1) activity (Park et al., 2022). Mechanistically, FLT3i inhibits mTOR and mTOR-dependent translation leading to cell death. However, in the presence of BM stromal factors, survival is restored despite FLT3i treatment by upregulating ATM/mTOR-dependent translation of essential oxidative phosphorylation (OXPHOS) genes. Moreover, we demonstrated that the combination of FLT3i and mTORC1 inhibition (mTORC1i) synergistically kills human FLT3-mutated AML cells in vitro and substantially reduces tumor burden and prevents relapse in mouse models transplanted with FLT3-mutated primary human AML. We observed that inhibition of mTOR alone has a minor effect on cell viability and a modest reduction in translation as measured by O-Propargyl Puromycin (OPP)-translation assays. Hence, to determine how these cells maintain translation and survival despite mTOR inhibition, we sought to identify the key factors mediating mTOR-independent translation. We tested roles for multiple kinases including CDK1, GSK3β, and p38, which have each been previously shown to phosphorylate 4E-BP1, in mTOR-independent translation in FLT3-mutated AML cells. We performed OPP assays to assess the impact of inhibitors against these kinases on translation activity in FLT3-mutated AML cell lines in the presence and absence of BM stromal factors. This approach revealed CDK1 as a critical regulator of mTOR-independent translation, suggesting that AML cells can maintain survival through this mTOR-independent pathway. Through immunoblotting, we demonstrate that a critical role for CDK1 in 4E-BP1 phosphorylation and protein translation in that combined inhibition of mTORC1 and CDK1 in FLT3-mutated AML cells leads to a near complete block in these activities even in the presence of BM stromal factors. Because of its canonical role in the cell cycle, we performed cell cycle assays in parallel with the OPP assays to assess the relevance of cell cycle state in CDK1-dependent translation. The results show that concentrations of CDK1 inhibitor that only modestly impact the cell cycle can still markedly inhibit translation. These studies define a new role of CDK1 as a critical regulator of mTOR-independent translation and thus survival of FLT3-mutated AMLs. Further investigation of CDK1-dependent translation will leverage transcriptome/ translatome analysis to identify which mRNAs are being specifically translated in mTOR-dependent versus CDK1-dependent manner to sustain FLT3-mutated AML survival against FLT3i-based therapies. Ongoing studies are also dissecting the mechanism whereby ATM promotes mTOR expression and activity. In all, understanding how translation and thus AML cell survival is maintained independently of mTOR is critical for evaluating efficacy of therapies targeting FLT3 and mTOR kinases, upfront AML resistance to these therapies, and the evolution of resistance.

Supplemental Materials and Methods

Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults with a five-year survival rate of ~30%. Approximately 30% of AML patients harbor an internal tandem duplication (ITD) mutation in the FMS related receptor tyrosine kinase 3 (FLT3) gene, which is an indicator of poor overall survival and a high relapse rate. These mutations cause constitutive activation of the FLT3 receptor, which amplify proliferation and survival signaling that supports leukemia progression and chemotherapy resistance. Although FLT3 inhibitors (FLT3i) improve median survival by a couple of months, and they have reduced toxicity relative to conventional chemotherapy in clinical trials, patients eventually acquire resistance and relapse. Clinical trials have revealed that AML blasts in the bone marrow (BM) were not effectively eliminated compared to AML blasts in peripheral blood, suggesting a protective role of the BM microenvironment that counteracts FLT3i therapy. A clear understanding of FLT3-mutated AML biology and how it is influenced by the microenvironment is imperative for developing new molecular approaches for improving efficacy of FLT3i-based therapies. We have discovered that BM stromal cell mediated regulation of Ataxia Telangiectasia Mutated (ATM) promotes Mammalian Target of Rapamycin Complex 1 (mTORC1) activity despite FLT3 inhibition, which maintains translation of oxidative phosphorylation genes that are critical for AML cell survival upon treatment with FLT3i [PMID: 36259537]. Key findings show that FLT3i inhibits mTOR and mTOR-dependent translation leading to cell death. However, in the presence of BM stromal factors, survival is restored upon FLT3i by regulating ATM/mTOR-dependent translation of essential phosphorylation genes to overcome FLT3i. Moreover, we demonstrated that the combination of FLT3i and mTORC1 inhibition (mTORC1i) synergistically kills human FLT3-mutated AML cells in vitro and substantially reduces tumor burden and prevents relapse in mouse models of FLT3-mutated AML. Further studies measuring translation in FLT3-mutated AML cells revealed that there are at least two different translation pathways dictated by FLT3. One of these pathways is mTOR-dependent, as expected, and the other is mTOR-independent. Additional studies have sought to characterize FLT3-controlled translation, independent of mTOR, and its importance for FLT3-mutated AML cell survival. Although highly sensitized to FLT3i, we observed that ATM/mTOR-deficient cells maintain near-normal translation and survival. Additionally, mounting evidence suggests that there are other kinases, such as CDK1, GSK3β, and p38, that drive mTOR-independent translation by phosphorylating 4E-BP1. With this knowledge, we took an unbiased approach and used O-Propargyl Puromycin (OPP)-translation assays to assess the impact of inhibitors against these kinases on translation activity in FLT3-mutated AML cell lines. This approach revealed CDK1 as a critical regulator of translation downstream of FLT3. Interestingly, myeloid progenitors reprogram to an mTOR-independent translational program requiring phosphorylation of 4E-BP1 by CDK1 [PMID: 32386556], which could be relevant for myeloid leukemias. Further corroborating my findings, RNA sequencing data suggest that FLT3 positively regulates CDK1 expression/activity, suggesting a downstream position in parallel with mTOR. In conclusion, we have uncovered a FLT3-controlled translation pathway that promotes FLT3-mutated AML cell survival independent of mTOR. Further characterization of this pathway could help predict mechanisms of resistance to FLT3 and mTOR inhibitors in AML and could identify new targets to promote more durable remissions in patients.

Figure S1. The glutaminase inhibitor CB-839 impairs glutathione metabolism in FLT3WT AML cells. Figure S2. The glutaminase inhibitor CB-839 decreases glutathione levels, but does not increase total cellular ROS levels in AML cells. Figure S3. Antioxidant vitamin E suppresses mitoROS and apoptosis induced by CB-839/pro-oxidant combination therapies. Figure S4. CB-839 cooperates with the pro-oxidant drug ATO in inducing apoptosis, mitoROS and AML cell death. Figure S5. CB-839/HHT therapy induces total cellular ROS. AML cells that survive therapy are metabolically similar to drug naive cells and are not resistant to subsequent therapy. Figure S6. CB-839/ATO and CB-839/HHT combination therapies do not exhibit toxicity in vivo in mice or toward normal human CD34+ cells. Figure S7. CB-839 cooperates with HHT in inducing mitoROS and apoptosis in primary human AML cells and cell death/apoptosis in ALL cells. Table S1. Combination index (CI values) for drug combinations tested in cell viability assays. Table S2. The clinical characteristics of the AML cohort studied are summarized.

Supplementary Methods - PDF file 124K, Supplementary Methods

Supplemental Methods. This supplementary file includes additional methods for sample preparation, Seahorse analysis of oxygen consumption rates, reactive oxygen species measurements, flow cytometry analysis, NMR analysis, RT-PCR conditions, quizartinib synthesis information.

Supplemental Figures 1-8. Figure 1. Mitochondrial metabolism becomes essential for TKI-treated BCR-ABL+ leukemia cells. Figure 2: Oligomycin-A sensitizes cells to BCR-ABL inhibition. Figure 3. Oligomycin-A sensitizes cells to BCR-ABL inhibition. Figure 4. Oligomycin-A sensitizes acute myeloid leukemia cells to TKI. Figure 5. Low nM concentrations of oligomycin-A do not perturb the TCA cycle. Figure 6. Oligomycin-A disrupts mitochondrial functions in leukemia cells. Figure 7. Low dose oligomycin-A synergizes with TKI to eliminate leukemia in vivo with no significant toxicity. Figure 8. Model for how TKI treatment of leukemias creates an altered metabolic state sensitive to mitochondrial perturbations.

Prior to the past few years, the development of new therapies for acute myeloid leukemia (AML) has been disappointingly slow. For several decades, the standard therapy for AML has consisted of intensive induction chemotherapy, and potentially a subsequent hematopoietic stem cell transplant. Unfortunately, older patients are less responsive to, and are frequently unfit to tolerate, such intensive chemotherapy. Given that a majority of AML patients are elderly, this population has been most affected by the lack of newer less toxic therapies. However, in recent years, the treatment landscape for AML has dramatically shifted with the approval of many new drugs. As summarized in this review, several of these new drugs are targeted agents that are better tolerated than standard chemotherapy and could substantially benefit elderly patients. Although drug resistance remains a major concern, the treatment options for elderly AML patients are more numerous than ever before, bringing new promise for improved patient outcomes.

Supplemental Tables 1-4. Table 1: pLKO shRNA clone information and antibody information. Table 2: Combination Indices to assess for synergism. Table 3: Quantitative 1H-, 31P- and 13C-NMR analysis of K562 CML cells treated with vehicle imatinib, oligomycin-A or the combination imatinib + oligomycin-A. Table 4: Complete blood counts and metabolic profiles after in vivo treatment with Oligomycin-A

The bone marrow (BM) microenvironment has been shown to promote drug resistance in leukemic cells. In the present study, we demonstrate that BM stromal cells induce alterations in gene expression and signaling pathways in FLT3-dependent acute myeloid leukemia (AML) cells that impact the efficacy of FLT3-targeted therapy. Specifically, we discovered that restoration of mTOR signaling by BM stromal factors mediates protection of AML cells from apoptosis following FLT3 inhibition through increased expression and selective translation of oxidative phosphorylation transcripts. Addition of an mTOR inhibitor to FLT3-targeted therapy not only significantly reduced leukemic burden in the BM in vivo, but also prevented relapse. We further determined that ATM (ataxia-telangiectasia mutated) plays an essential role in BM stroma-mediated survival as a key upstream regulator of mTOR signaling. These studies reveal how the BM microenvironment promotes survival of AML cells in the face of FLT3 inhibition through maintenance of oxidative phosphorylation. SIGNIFICANCE Our results uncover a novel pathway activated by the BM microenvironment that provides protection of AML cells from therapeutic elimination following FLT3 inhibition. These studies identify candidates for combinatorial therapies designed to overcome the protective effects of BM and improve outcomes for AML patients.

Accurate cell typing is fundamental to analysis of spatial single-cell transcriptomics, but legacy scRNA-seq algorithms can underperform in this new type of data. We have developed a cell typing algorithm, Insitutype, designed for statistical and computational efficiency in spatial transcriptomics data. Insitutype is based on a likelihood model that weighs the evidence from every expression value, extracting all the information available in each cell’s expression profile. This likelihood model underlies a Bayes classifier for supervised cell typing, and an Expectation-Maximization algorithm for unsupervised and semi-supervised clustering. Insitutype also leverages alternative data types collected in spatial studies, such as cell images and spatial context, by using them to inform prior probabilities of cell type calls. We demonstrate rapid clustering of millions of cells and accurate fine-grained cell typing of kidney and non-small cell lung cancer samples.

While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance toward improving therapeutic outcomes. In this study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn upregulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance and a promising strategy for improved treatment of FLT3 mutant AML patients.

Internal tandem duplication (ITD) mutations in FMS-like tyrosine kinase 3 (FLT3) are among the most common mutations in acute myeloid leukemia (AML) and are associated with poor prognosis. FLT3-ITD causes constitutive activation of FLT3, and the strong evidence that activated FLT3 drives leukemogenesis has led to the development of several FLT3-targeted inhibitors. However, clinical studies of FLT3-targeted inhibitors have demonstrated much more effective clearing of leukemic burden in the periphery than in the bone marrow, implicating the bone marrow microenvironment as a potential contributor to drug resistance. As previously reported, the conditioned media of human bone marrow stromal cells (CM-BMSC) protects human FLT3-ITD AML cells from the killing effect of FLT3-targeted therapy despite complete inhibition of FLT3. We further show that genetic knockdown of Ataxia Telangiectasia Mutated (ATM) in combination with FLT3 inhibition substantially reverses the protection from cell death mediated by CM-BMSC. While autophosphorylation activity of ATM is downregulated upon FLT3 inhibition in regular media, CM-BMSC prevented the loss of ATM activity. Interestingly, genetic knockdown of ATM in human FLT3-ITD AML cells impaired the activity of mTOR Complex 1, and unbiased analyses of gene expression and signaling pathways by RNA-seq analysis and Reverse Phase Protein Arrays (RPPA) showed that mTOR signaling is downregulated with FLT3 inhibition in regular media, coinciding with marked inhibition of the protein translation machinery. Both mTOR signaling and protein translation were maintained during FLT3 inhibition in the presence of CM-BMSC. Furthermore, human FLT3-ITD AML cells treated with mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib reversed the protection mediated by CM-BMSC, and combination therapies are currently being tested in mouse models. These data suggest that ATM kinase and mTOR signaling play a key role in bone marrow stoma-mediated protection against FLT3 targeted therapy. Findings from this research provide new insights into the mechanism of bone marrow stroma-mediated protection of FLT3-ITD AML from FLT3-targeted therapy, identifying additional candidates for combinatorial therapies designed to overcome the protective effects of bone marrow stromal cells and improve patient outcomes. Citation Format: Hae J. Park, Mark A. Gregory, Vadym Zaberezhnyy, James DeGregori. Targeting ATM kinase and mTOR signaling reverses bone marrow stromal cell-mediated protection of FLT3-ITD AML from FLT3-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1442.

BackgroundAlthough cancer immunotherapies can effectively restore T cell-mediated immunity leading to sustained clinical responses, these responses are unpredictable partly due to highly heterogeneous phenotypes of tumor-infiltrating lymphocytes (TILs) between patients. Thus, understanding such TILs and their roles in the context of tumor microenvironments (TME) may lead to developing better immunotherapy solutions. The spatial molecular imager (SMI) is a novel spatial transcriptomics platform that allows spatially resolved high-dimensional cellular phenotyping for comprehensive TIL profiling. SMI uses fluorescent molecular barcodes to enable in-situ measurement of biological targets on an intact tissue sample. Here, we characterize comprehensive TIL phenotypes and visualize landscape of TILs directly on intact formalin-fixed paraffin-embedded (FFPE) tissues using a 1000+-plex RNA panel.MethodsTo build multi-omics TIL profiling data sets for renal cell carcinoma (RCC) tissues, we employed scRNA-seq, mass cytometry (CyTOF) and SMI. Peripheral blood mononuclear cells and dissociated cells from matched RCC tumor and adjacent normal tissues were analyzed by CyTOF and single-cell sequencing. Then, SMI profiling of matching FFPE tissues was used to visualize TILs in the context of the TME and to understand relationships between high-dimensional cellular heterogeneity and the spatial organization of cells within a tumor tissue.ResultsCyTOF and scRNA-seq analysis of dissociated cells was used to determine the gene expression profiles of numerous cellular subsets. TCR sequencing was also used to assess the extent of clonal expansion and clonotypic relationships between blood and tumor. Consistent with our previous reports, T cell populations could be segregated based on markers associated with chronic T cell receptor signaling and many T cells with an exhausted phenotype were clonally expanded in the tumor but not the blood. In contrast, T cell clonotypes with bystander phenotypes in the tumor were readily detected as expanded clones in the blood, supporting notion that not all tumor-infiltrating T cells are specific for tumor antigens. SMI analysis of matched tumor tissue was used to accurately quantify the densities and to determine the spatial organization of all T cell subsets. In addition, computational methods were used to describe distinct cellular niches within tumors with accurately defined cellular compositions.ConclusionsHigh dimensional cellular profiling highlights the abundance of bystander T cell infiltration of RCC tumors. Comprehensive spatial profiling by SMI provides spatial context to the highly diverse immune cell composition of tumor infiltrates.Ethics ApprovalFully anonymous human material was obtained from Northwest Biotrust and given IRB designation of non-human subjects research.

Abstract Purpose: Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional chemotherapy and a diagnosis of AML is usually fatal. More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. These studies were directed at determining the effects of glutaminase inhibition on metabolism in AML and identifying general weaknesses that can be exploited therapeutically. Experimental Design: AML cancer cell lines, primary AML cells, and mouse models of AML and acute lymphoblastic leukemia (ALL) were utilized. Results: We show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples, and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating ALL cells in vitro and in vivo. Conclusions: Targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia.

Acute myeloid leukemia (AML) is a blood cancer that is poorly responsive to conventional cytotoxic chemotherapy and a diagnosis of AML is usually fatal. More effective and better-tolerated therapies for AML are desperately needed. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are one of the most frequently observed genetic defects in AML. FLT3 inhibitors have shown impressive anti-leukemic activity in clinical trials; however, sustained remissions using these inhibitors as monotherapy have not been achieved. Our previous studies have implicated impaired glutamine metabolism in response to FLT3 inhibitors as a dominant factor causing AML cell death. In this study, we have employed metabolic flux analysis to examine the effects of FLT3 inhibition on glutamine utilization in FLT3-mutated AML cells using stable isotope tracers. We found that the FLT3 inhibitor AC220 inhibited glutamine flux into the antioxidant factor glutathione profoundly due to defective glutamine import. We also found that the glutaminase inhibitor CB-839 similarly impaired glutathione production by effectively blocking flux of glutamine into glutamate. Moreover, the combination of AC220 with CB-839 synergized to deplete glutathione, induce mitochondrial reactive oxygen species, and cause loss of viability through apoptotic cell death. In vivo, glutaminase inhibition with CB-839 facilitated leukemic cell elimination by AC220 and improved survival significantly in a patient-derived xenograft AML mouse model. Therefore, targeting glutaminase in combination with FLT3 may represent an effective therapeutic strategy for improving treatment of FLT3-mutated AML.

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

Abstract Acute myeloid leukemia (AML) is the most common adult acute leukemia and accounts for approximately 20% of childhood leukemias. Although frontline treatment of AML with cytotoxic chemotherapy is capable of achieving high remission rates, 75-80% of patients will either not respond to or will relapse after initial therapy, and most patients will die of their disease. More effective and better-tolerated therapies for AML are required. Activating mutations in fms-like tyrosine kinase 3 (FLT3) are the most frequently observed genetic defect in AML and drive leukemic cell growth and survival. FLT3 tyrosine kinase inhibitors have shown impressive anti-leukemic activity in clinical trials, however, sustained remissions using these inhibitors as monotherapy have not been achieved. In order to identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a large-scale RNA interference-based screen. The screen identified several genes involved in metabolic regulation, including ataxia telangiectasia mutated (ATM), as being synthetic lethal with FLT3 inhibition in FLT3 mutated AML. Genetic or pharmacological inactivation of ATM or its downstream effector glucose-6-phosphate dehydrogenase (G6PD) sensitized AML cells to FLT3 inhibition through enhancing apoptosis. Whole metabolome profiling revealed that FLT3 inhibition causes severe and widespread metabolic deficiencies, including depletion of the antioxidant factor glutathione. Inactivation of either ATM or G6PD exacerbated glutathione depletion upon FLT3 inhibition. Subsequent analyses revealed that FLT3 inhibition elicits severe mitochondrial oxidative stress that is causative in apoptosis and accentuated by ATM or G6PD inhibition. The use of a drug that promotes the production of mitochondrial reactive oxygen species (ROS) in combination with a FLT3 inhibitor augmented elimination of AML cells both in vitro and in vivo. Our data support the hypothesis that FLT3 mutated AML cells are highly dependent on FLT3 activity to maintain glutathione levels and sufficient mitochondrial antioxidant capacity to sustain cell survival. Moreover, these data support the novel strategy of employing mitochondrial ROS inducing agents as adjuvant to FLT3 inhibitor therapy to more effectively treat FLT3 mutated AML. Citation Format: Mark A. Gregory, Angelo D'Alessandro, Francesca Alvarez-Calderon, Jihye Kim, Travis Nemkov, Aik Choon Tan, Kirk C. Hansen, James DeGregori. ATM/G6PD-dependent metabolic pathways promote mitochondrial redox homeostasis and resistance to FLT3 inhibition in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C78.