Human B-cell Acute Lymphoblastic Leukemia Cell Research Articles

Chemotherapy-initiated cysteine-rich protein 61 decreases acute B-lymphoblastic leukemia chemosensitivity

PurposeChemoresistance is a major challenge for acute lymphoblastic leukemia (ALL) treatment. Cysteine-rich protein 61 (Cyr61) plays an important role in drug resistance modulation of tumor cells, and Cyr61 levels are increased in the bone marrow of patients with ALL and contribute to ALL cell survival. However, the effect of Cyr61 on B cell acute lymphoblastic leukemia (B-ALL) cell chemosensitivity and the regulatory mechanisms underlying Cyr61 production in bone marrow remain unknown.MethodsNalm-6 and Reh human B-ALL cell lines were used in this study. Cyr61 levels were assessed using quantitative real-time PCR (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay. The effect of Cyr61 on B-ALL cell chemosensitivity to daunorubicin (DNR) was evaluated using cell viability and flow cytometry analyses. The regulatory mechanisms of Cyr61 production in bone marrow were examined using qRT-PCR and western blot analysis.ResultsCyr61 knockdown and overexpression increased and decreased the chemosensitivity of B-ALL cells to DNR, respectively. Cyr61 attenuated chemotherapeutic drug-induced apoptosis by upregulating B cell lymphoma-2. Notably, DNR induced DNA damage response and increased Cyr61 secretion in B-ALL cells through the ataxia telangiectasia mutated (ATM)-dependent nuclear factor kappa B pathway.ConclusionDNR induces Cyr61 production in B-ALL cells, and increased Cyr61 levels reduce the chemosensitivity of B-ALL cells. Consequently, targeting Cyr61 or related ATM signaling pathway may present a promising treatment strategy to enhance the chemosensitivity of patients with B-ALL.

PTEN Counter Balances mTORC1 and PI3K Activities to Maintain Metabolic Homeostasis of B-ALL

Significance PIK3CA activating mutations and PTEN deletions are frequent in nearly all types of cancer but hardly detected in B cell acute lymphoblastic leukemia (B-ALL). Our study revealed unexpected energy crisis in terms of ATP deficit and oxidative phosphorylation (OXPHOS) impairment in Pten depleted B-ALL cells. Strikingly, a significant reduction of MYC levels as well as cell viability were observed in both murine and human B-ALL cells with PI3K hyperactivation. Furthermore, forced MYC expression not only recovered cell survival but also lifted restriction of mTORC1 signaling in Pten deficient B-ALL cells. Here we reported unique crosstalk of PI3K and mTORC1 activities which is mediated by MYC to ensure balanced cellular energy supply and anabolism in B-ALL cells. Results We revealed the essentiality of PTEN in ALL cells was through keeping energy production by glutaminolysis. Seahorse analysis and glutamine consumption assay showed significant half-fold reduction of both oxygen consumption rate and glutamine utilization in Pten KO ALL cells. Importantly, these defects were fully rescued by ectopic expression of MYC which drastically increased cell mass and proliferation of Pten KO B-ALL cells. By overexpressing the Myc T58A mutant, which is resistant to proteasomal degradation, we found a further 2-fold enhancement of cell growth compared to wild-type Myc in Pten KO ALL cells. We further demonstrated that the half-life of Myc in Pten KO cells was significantly shortened through proteasomal degradation, suggesting the loss of Pten disrupted Myc stability. Based on the transcriptomic profiling of Pten deficient ALL cells, we found that PI3K hyperactivation decreased multiple cellular biosynthetic processes, while Myc overexpression significantly restored biosynthesis, indicating a potential role of Myc in preserving anabolic activity. Untargeted metabolomic analysis indicated depletion of amino acid levels in Pten KO murine B-ALL cells, while Myc overexpression partially restored these levels. We further demonstrated arginine, one of the amino acids that sensed by mTORC1, was significantly enriched in MYC overexpressed Pten KO ALL cells and mediated mTORC1 full activation. Those results indicate the pivotal function of Myc in synergizing PI3K and mTORC1 signaling to promote oncogenic metabolism of B-ALL. Conclusion In summary, our study revealed the essential function of PTEN in maintaining cellular metabolic homeostasis through antagonizing PI3K hyperactivation in B-ALL. We further demonstrated that MYC levels was leveraged by PI3K signaling to prevent mTORC1 activation and further biosynthetic elevation. PI3K hyperactivation was found to deplete energy reserve and restrain biomass accumulation, which destructs metabolic adaptation to oncogenic proliferation in B-ALL cells. Our findings decipher a unique synthetic vulnerability of PI3K and MYC in malignant precursor B cells and highlight the balance between anabolism and energy supply for cell survival as a promising therapeutic strategy for B-ALL.

MiR-126 identifies a quiescent and chemo-resistant human B-ALL cell subset that correlates with minimal residual disease.

Complete elimination of B-cell acute lymphoblastic leukemia (B-ALL) by a risk-adapted primary treatment approach remains a clinical key objective, which fails in up to a third of patients. Recent evidence has implicated subpopulations of B-ALL cells with stem-like features in disease persistence. We hypothesized that microRNA-126, a core regulator of hematopoietic and leukemic stem cells, may resolve intratumor heterogeneity in B-ALL and uncover therapy-resistant subpopulations. We exploited patient-derived xenograft (PDX) models with B-ALL cells transduced with a miR-126 reporter allowing the prospective isolation of miR-126(high) cells for their functional and transcriptional characterization. Discrete miR-126(high) populations, often characterized by MIR126 locus demethylation, were identified in 8/9 PDX models and showed increased repopulation potential, in vivo chemotherapy resistance and hallmarks of quiescence, inflammation and stress-response pathway activation. Cells with a miR-126(high) transcriptional profile were identified as distinct disease subpopulations by single-cell RNA sequencing in diagnosis samples from adult and pediatric B-ALL. Expression of miR-126 and locus methylation were tested in several pediatric and adult B-ALL cohorts, which received standardized treatment. High microRNA-126 levels and locus demethylation at diagnosis associate with suboptimal response to induction chemotherapy (MRD > 0.05% at day +33 or MRD+ at day +78).

Histone acetylation by HBO1 (KAT7) activates Wnt/β-catenin signaling to promote leukemogenesis in B-cell acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological disorder with a dismal prognosis. The dysregulation of histone acetylation is of great significance in the pathogenesis and progression of B-ALL. Regarded as a fundamental acetyltransferase gene, the role of HBO1 (lysine acetyltransferase 7/KAT7) in B-ALL has not been investigated. Herein, we found that HBO1 expression was elevated in human B-ALL cells and associated with poor disease-free survival. Strikingly, HBO1 knockdown inhibited viability, proliferation, and G1-S cycle progression in B-ALL cells, while provoking apoptosis. In contrast, ectopic overexpression of HBO1 enhanced cell viability and proliferation but inhibited apoptotic activation. The results of in vivo experiments also certificated the inhibitory effect of HBO1 knockdown on tumor growth. Mechanistically, HBO1 acetylated histone H3K14, H4K8, and H4K12, followed by upregulating CTNNB1 expression, resulting in activation of the Wnt/β-catenin signaling pathway. Moreover, a novel small molecule inhibitor of HBO1, WM-3835, potently inhibited the progression of B-ALL. Our data identified HBO1 as an efficacious regulator of CTNNB1 with therapeutic potential in B-ALL.

The 'omics of obesity in B-cell acute lymphoblastic leukemia.

The obesity pandemic currently affects more than 70 million Americans and more than 650 million individuals worldwide. In addition to increasing susceptibility to pathogenic infections (eg, SARS-CoV-2), obesity promotes the development of many cancer subtypes and increases mortality rates in most cases. We and others have demonstrated that, in the context of B-cell acute lymphoblastic leukemia (B-ALL), adipocytes promote multidrug chemoresistance. Furthermore, others have demonstrated that B-ALL cells exposed to the adipocyte secretome alter their metabolic states to circumvent chemotherapy-mediated cytotoxicity. To better understand how adipocytes impact the function of human B-ALL cells, we used a multi-omic RNA-sequencing (single-cell and bulk transcriptomic) and mass spectroscopy (metabolomic and proteomic) approaches to define adipocyte-induced changes in normal and malignant B cells. These analyses revealed that the adipocyte secretome directly modulates programs in human B-ALL cells associated with metabolism, protection from oxidative stress, increased survival, B-cell development, and drivers of chemoresistance. Single-cell RNA sequencing analysis of mice on low- and high-fat diets revealed that obesity suppresses an immunologically active B-cell subpopulation and that the loss of this transcriptomic signature in patients with B-ALL is associated with poor survival outcomes. Analyses of sera and plasma samples from healthy donors and those with B-ALL revealed that obesity is associated with higher circulating levels of immunoglobulin-associated proteins, which support observations in obese mice of altered immunological homeostasis. In all, our multi-omics approach increases our understanding of pathways that may promote chemoresistance in human B-ALL and highlight a novel B-cell-specific signature in patients associated with survival outcomes.

Cmpd10357 to treat B-cell acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of cancer found in children. Although the overall survival rates are now >80%, 15%-20% of pediatric patients relapse, with survival rates subsequently dropping to 5%-10%. Cmpd10357, 3-amino-5-arylamino-6-chloro-N- (diaminomethylene) pyrazine-2-carboximide, is a highly potent, cell-permeant compound recently shown to have cytotoxic effects on solid tumors, including human breast cancer and high-grade gliomas, independent of their proliferative status. Cmpd10357 demonstrated concentration-dependent cytotoxicity in two human B-ALL cell lines, JM1 and Reh, at half-maximal inhibitory concentrations (IC50) of 3.2 and 3.3 μM, respectively. Cmpd10357, at a dose of 5 mg/kg, significantly prolonged survival in our B-ALL xenograft mouse model, with a median survival time of 49.0 days compared with 45.5 days in the control group (p < 0.05). The cytotoxicity of Cmpd10357 demonstrated caspase-independent, nonapoptotic cancer cell demise associated with the nuclear translocation of apoptosis-inducing factor (AIF). The cytotoxicity of Cmpd10357 in B-ALL cells was inhibited by Necrostatin-1 but not by Necrosulfonamide. These studies suggest that an AIF-mediated, caspase-independent necrosis mechanism of Cmpd10357 in B-ALL could be used in combination with traditional apoptotic chemotherapeutic agents.

Abstract 846: Transcriptional regulation of Aurora-B kinase expression in B-cell acute lymphoblastic leukemia (B-ALL)

Abstract The Aurora kinases are serine/threonine kinases that are essential for mitosis. Aurora-B kinase is a component of the chromosomal passenger complex (CPC), chromosome condensation, the spindle-assembly checkpoint, and for cytokinesis. The activity of Aurora-B is essential for cellular division and proliferation. Inhibition of Aurora-B is cytotoxic, and inhibitor of Aurora-B - Barasertib (AZD1152) has been tested in clinical trials against different types of malignancies, including leukemia. However, regulation of Aurora-B expression is still largely unknown. Here, we present evidence that expression of Aurora-B in B-cell acute lymphoblastic leukemia (B-ALL) is regulated at the transcriptional level by Ikaros, a transcription factor and tumor suppressor protein. Analysis of global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) in several B-ALL primary human B-ALL cells and cell lines, showed a strong enrichment of Ikaros at the promoter of the Aurora-B gene. Ikaros functions as a tumor suppressor protein and deletion of Ikaros is associated with development of high-risk B-ALL. Ikaros binding to Aurora-B promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary B-ALL cells. The role of Ikaros in regulating Aurora-B transcription in B-ALL was tested using gain-of-function and loss-of-function experiments. Overexpression of Ikaros in human B-ALL was associated with strongly reduced transcription and overall expression of Aurora-B. Ikaros knock-down with shRNA results in increased expression of Aurora-B in B-ALL. Since Ikaros function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of Aurora-B in B-ALL. Overexpression of CK2 via retroviral transduction resulted in increased expression of the Aurora-B gene. Increased expression of CK2 was associated with a loss of Ikaros binding to the Aurora-B gene promoter. Molecular inhibition of CK2 using shRNA resulted in reduced expression of Aurora-B in human B-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the Aurora-B promoter. Ikaros knock-down abolished downregulation of Aurora-B expression in B-ALL cells following treatment with CK2 inhibitors. These data demonstrate that CK2 and Ikaros are critical regulators of Aurora-B expression in B-ALL. In conclusion, these results indicate that expression of the Aurora-B gene in B-ALL is regulated by the CK2-Ikaros signaling axis and provide a novel insight into mechanisms that regulate mitosis in leukemia. Citation Format: Yali Ding, Chunhua Song, Joseph Schramm, Soumya Maru, Daniel Bogus, Dhimant H. Desai, Arati K. Sharma, Chandrika Gowda, Sinisa Dovat. Transcriptional regulation of Aurora-B kinase expression in B-cell acute lymphoblastic leukemia (B-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 846.

Interleukin-9 Promotes Chemoresistance in B-ALL Which Correlates with Decreased Bim and c-Raf Protein Expression

Background: According to the National Cancer Institute, B-cell acute lymphoblastic leukemia (B-ALL) is the most common cancer of children and adolescents (ALL, NCI, PDQ, accessed 8/2/2021). Recently, obesity has been identified as a risk factor which is associated with poor survival outcomes (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012) which is concerning due to the obesity rates in children and adolescents having tripled since the 1970's (Ogden et al., 2006; Ogden et al., 2020). Indeed, survival rates in obese pediatric patients with B-ALL can decline by as much as 30% relative to outcomes observed in lean patients, with obese patients more commonly presenting with treatment-related adverse events (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012). A hallmark of obesity is the accumulation of adipocytes, an endocrine cell type which can promote chemoresistance (Ehsanipour et al., 2013; Sheng et al., 2016; Mittelman., 2021). The mechanistic understanding of how adipocytes promote chemoresistance in B-ALL is still under investigation and further insight into this relationship could lead to the rational design of effective therapeutic strategies for obese patients with limited treatment options.Methods: A cytokine/chemokine array was performed on adipocyte and stromal cell secretomes to identify potential adipocyte-secreted inflammatory mediators, which may promote chemoresistance in human B-ALL cells. Once candidate cytokines were identified, we performed in vitro assays to measure how the addition or neutralization of proteins of interest impacted the proliferation, activation of signaling pathways, steady-state mitochondrial protein levels, and survival of human B-ALL cells in the absence or presence of chemotherapy treatment. Additionally, we mined publicly available databases to determine how protein-coding genes of interest were associated with patient survival. Furthermore, we have used the diet-induced murine model of obesity to determine how targeting candidate cytokines impact B-ALL pathogenesis.Results: We have made the novel finding that interleukin-9 (IL-9) levels are higher in adipose-rich microenvironments and activates pro-survival pathways that promote chemoresistance in human B-ALL cells. We have found that obese mice lacking IL-9 are more resistant to B-ALL development due to significant increases in survival outcomes compared to lean mice transplanted with B-ALL cells. Furthermore, we have discovered that human B-ALL cells upregulate the interleukin-9 receptors (IL-9R) when exposed to the adipocyte secretome. This potential feedback loop may increase the responsiveness of leukemia cells to local IL-9 levels. These observations were supported by our data mining results, which revealed that IL-9R gene expression levels were higher in more aggressive subtypes of B-ALL, including Ph-like B-ALL. When human B-ALL cells were treated with recombinant IL-9 (rIL-9), chemoresistance to methotrexate and doxorubicin was observed. Mechanistically, rIL-9 treatment of human B-ALL cells also downregulated the protein expression of the pro-apoptotic mitochondrial-associated protein Bim and pro-proliferative protein Raf. In all, our experiments have identified IL-9 as an adipocyte-enriched cytokine, which promotes pan-chemoresistance in human B-ALL cells. Furthermore, we have shown that this effect maybe mediated in part by suppressing the protein of expression of pro-apoptotic and proliferative proteins.Conclusions: To our knowledge, our results represent the first reports of IL-9 mediated chemoresistance in human B-ALL and the first to demonstrate that IL-9 regulates the protein homeostasis of anti- and pro-apoptotic mitochondrial proteins. In ongoing studies, we are conducting in vitro and murine studies with parental and IL-9R-deficient B-ALL cells to determine how B-ALL pathogenesis and chemosensitivity are impacted. Subsequent studies will be conducted in lean and obese mice transplanted with B-ALL cells who receive chemotherapy treatment alone or in combination with IL-9 neutralizing antibody administration. DisclosuresLee: PureTech Health: Research Funding. Henry: PureTech Health: Research Funding.

Adipocyte-secreted Interleukin-9 Promotes Leukemia Development and Chemoresistance to Front-line Chemotherapies

Abstract Background Obese pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) have inferior overall survival outcomes compared to lean patients, with obese patients having a 2.5-fold increased risk of an adverse event and an almost 4-fold greater risk of death. Emerging data suggest that the presence of factors in the obese microenvironment may directly promote chemoresistance in B-ALL cells. Objective Given the increasing prevalence of obesity and negative impact of obesity on cancer, in this study, we sought to determine how adipocytes impact B-ALL progression and responses to front-line chemotherapies. Results We found that circulating IL-9 levels were significantly elevated in obese mice fed high-fat diets compared to mice fed control diets. Furthermore, treating human B-ALL cells with recombinant IL-9 (rIL-9) promoted cell cycle progression and proliferation. Interestingly, rIL-9 stimulation of human B-ALL cells activated survival pathways, which coincide with the induction of chemoresistance to methotrexate and doxorubicin. Conclusions Our findings demonstrate that IL-9 levels are elevated in obese microenvironments and that this cytokine promotes B-ALL pathogenesis. In ongoing studies, we are determining how antibody-mediated neutralization of IL-9 impacts disease progression and chemosensitivity in B-ALL cells.

Obese microenvironments attenuate the efficacy of endogenous and CD19-directed CAR T-cells

Abstract Background: Hematological malignancies represent the largest class of cancers in pediatric patients. Despite successes in treating primary diseases, patients with relapsed B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL) have significantly lower survival outcomes. This has led to the use of immunotherapies, notably chimeric antigen receptor (CAR) T-cells, to treat relapse. Unfortunately, only 50% respond to CAR-T therapy, with relapse common in less than a year. Objective: Given that obesity is associated with poorer survival outcomes in pediatric patients with B-cell malignancies and promotes a loss of immune homeostasis, we hypothesize that obesity compromises the function of endogenous and engineered T-cells in B-cell malignancies. Results: Using in vitro models of adiposity and diet-induced obesity mouse models to study the impact of obesity on primary T-cell function, we observe defects in proliferation, cytokine production, and the upregulation of cytolytic mediators when cultured in adipocyte but not in bone marrow cell or unconditioned media. The introduction of CD19-direct CARs did not overcome adipocyte-induced defects in T-cell function, which resulted in increased PD-1 surface expression, compromised cytokine production, and defective killing of human CD19-expressing B-ALL cells. Interestingly, human B-ALL cells cultured in adipocyte-secreted factors downregulated CD19, suggesting that adipocytes promote B-ALL pathogenesis by compromising T-cell function and reducing the surface expression of antigens on target cells. Conclusion: Our studies are the first to demonstrate the inhibitory impact of the adipocyte secretome on CAR T-cell function in models of B-ALL.

Oxidative phosphorylation enhances the leukemogenic capacity and resistance to chemotherapy of B cell acute lymphoblastic leukemia.

How metabolic status controls the fates of different types of leukemia cells remains elusive. Using a SoNar-transgenic mouse line, we demonstrated that B cell acute lymphoblastic leukemia (B-ALL) cells had a preference in using oxidative phosphorylation. B-ALL cells with a low SoNar ratio (SoNar-low) had enhanced mitochondrial respiration capacity, mainly resided in the vascular niche, and were enriched with more functional leukemia-initiating cells than that of SoNar-high cells in a murine B-ALL model. The SoNar-low cells were more resistant to cytosine arabinoside (Ara-C) treatment. cyclic adenosine 3',5'-monophosphate response element-binding protein transactivated pyruvate dehydrogenase complex component X and cytidine deaminase to maintain the oxidative phosphorylation level and Ara-C-induced resistance. SoNar-low human primary B-ALL cells also had a preference for oxidative phosphorylation. Suppressing oxidative phosphorylation with several drugs sufficiently attenuated Ara-C-induced resistance. Our study provides a unique angle for understanding the potential connections between metabolism and B-ALL cell fates.

PON2 subverts metabolic gatekeeper functions in B cells to promote leukemogenesis

Unlike other cell types, developing B cells undergo multiple rounds of somatic recombination and hypermutation to evolve high-affinity antibodies. Reflecting the high frequency of DNA double-strand breaks, adaptive immune protection by B cells comes with an increased risk of malignant transformation. B lymphoid transcription factors (e.g., IKZF1 and PAX5) serve as metabolic gatekeepers by limiting glucose to levels insufficient to fuel transformation. We here identified aberrant expression of the lactonase PON2 in B cell acute lymphoblastic leukemia (B-ALL) as a mechanism to bypass metabolic gatekeeper functions. Compared to normal pre-B cells, PON2 expression was elevated in patient-derived B-ALL samples and correlated with poor clinical outcomes in pediatric and adult cohorts. Genetic deletion of Pon2 had no measurable impact on normal B cell development. However, in mouse models for BCR-ABL1 and NRASG12D-driven B-ALL, deletion of Pon2 compromised proliferation, colony formation, and leukemia initiation in transplant recipient mice. Compromised leukemogenesis resulted from defective glucose uptake and adenosine triphosphate (ATP) production in PON2-deficient murine and human B-ALL cells. Mechanistically, PON2 enabled glucose uptake by releasing the glucose-transporter GLUT1 from its inhibitor stomatin (STOM) and genetic deletion of STOM largely rescued PON2 deficiency. While not required for glucose transport, the PON2 lactonase moiety hydrolyzes the lactone-prodrug 3OC12 to form a cytotoxic intermediate. Mirroring PON2 expression levels in B-ALL, 3OC12 selectively killed patient-derived B-ALL cells but was well tolerated in transplant recipient mice. Hence, while B-ALL cells critically depend on aberrant PON2 expression to evade metabolic gatekeeper functions, PON2 lactonase activity can be leveraged as synthetic lethality to overcome drug resistance in refractory B-ALL.

Obesity-Induced Chemoresistance in B-Cell Acute Lymphoblastic Leukemia Cells Is Mediated By Reduced Chemotherapy Uptake and Interleukin-9 Signaling

Background: Since the 1970's, obesity rates in children and adolescents have more than tripled (Ogden et al., 2006; Ogden et al., 2016) which parallels the rise in annual medical cost to treat obese pediatric patients in the United States which now exceeds $10 billion dollars (Beiner et al., 2020). The increasing cost to treat obese pediatric patients is in part due to more aggressive diseases which frequently present in these patients. For example, obese pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) have inferior overall survival compared to lean patients, with obese patients having a 2.5-fold increased risk of developing an adverse event and an almost 4-fold greater risk of death compared to non-obese patients (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012). Adipocytes accumulate with weight gain, and emerging data suggest the these cells secrete factors which promote chemoresistance in B-ALL cells (Ehsanipour et al., 2013; Sheng et al., 2016). Given the negative impact of obesity on cancer outcomes, we sought to define how adiposity impacts B-ALL pathogenesis and chemosensitivity. Methods: We performed global cytokine profiling and ELISA analyses to identify cytokines and chemokines secreted at high levels by adipocytes and present at high concentrations in the obese relative to the lean microenvironments. We then determined how candidate cytokines/chemokines impacted the function (proliferation/ signal transduction) of murine and human B-ALL cell lines using in vitro models of adiposity and the diet-induced murine model of obesity (DIO model). These experiments were also carried out on de-identified patient samples from lean and obese pediatric patients with B-ALL which were obtained from the Aflac Leukemia and Lymphoma Biorepository. To assess the impact of bone marrow stromal cells and adipocytes on B-ALL cell chemosensitivity, we exposed human B-ALL cells to stromal cell conditioned-media (SCM) and adipocyte-conditioned media (ACM) prior to methotrexate (MTX) treatment to determine their impact on cytotoxicity (Annexin-V/PI analysis) and drug uptake (confocal microscopy using fluorescently-conjugated methotrexate MTX). Results: We made the novel finding that Interleukin-9 is elevated in adipose-rich microenvironments and induces survival pathways which promote chemoresistance. From our studies, we found that obese mice succumb significantly faster than lean mice transplanted with murine Ph+ B-ALL cells. Analysis of serum obtained from lean and obese mice revealed that circulating IL-9 levels were significantly higher in mice with high adiposity. Furthermore, we found that adipocytes secreted copious amounts of IL-9; whereas, the production of this cytokine by bone marrow stromal cells was barely detectable. Interestingly, only human B-ALL cells exposed to ACM upregulated the Interleukin-9 receptor, suggesting increased sensitivity to this cytokine when malignant cells are exposed to adipocytes. Treating human B-ALL cells with recombinant IL-9 resulted in the activation of STAT3 and NF-κB signaling pathways and significantly increased proliferation. The induction of these proliferative and survival pathways coincided with reduced sensitivity to MTX treatment, whereas B-ALL cell death was abrogated when malignant cells were exposed to high levels of IL-9. Furthermore, the uptake of fluorescently-labeled MTX in ACM-exposed human B-ALL cells was significantly decreased compared to the internalization observed in B-ALL cells cultured in unconditioned media or SCM. Conclusions:It has been previously shown that IL-9 promotes chemoresistance in diffuse large B-cell lymphoma and is associated with worse prognosis in patients with B-cell chronic lymphocytic leukemia (Chen, Lv, Li, Lu, &amp; Wang, 2014; Lv, Feng, Ge, Lu, &amp; Wang, 2016). To our knowledge, our studies are the first to demonstrate that adipocyte-produced IL-9 promotes B-ALL pathogenesis and chemoresistance. To further explore this relationship and potential of targeting IL-9 as a novel therapeutic option, we are conducting in vitro and in vivo experiments to assess the impact of IL-9 neutralizing antibody treatment alone and when combined with frontline chemotherapy options for B-ALL Disclosures No relevant conflicts of interest to declare.

Obesity Attenuates T-Cell Function Which Impacts the Efficacy of T-Cell Based Immunotherapies in Acute Lymphoblastic Leukemia

The incidence of obesity continues to rise with over 50% of the world's population predicted to be overweight/obese by the year 2030. The global health impact of this trend is concerning given that obesity is a risk factor for developing cancers of varying etiologies. Alarmingly, the survival outcomes for obese patients with cancer are lower than those observed in lean patients. Obesity is characterized by the accumulation of adipocytes which alters drug dynamics and impacts the function of cancer and immune cells in the tumor microenvironment. Obesity-induced immune defects are troubling given the increasing use of immunotherapy in the treatment of malignancies. Here we show that adipocyte-secreted factors upregulate immunosuppressive mechanisms on human B-cell acute lymphoblastic leukemia (B-ALL) cells, attenuate the function of endogenous T-cells, and compromise the efficacy of T-cell based immunotherapies. To study the impact of adiposity on T-cell function, CD4+ and CD8+ T-cells were purified from the spleens of C57BL/6 mice and activated with PMA/Ionomycin for 72 hours in unconditioned media (UCM), bone marrow stromal-cell conditioned media (SCM), and adipocyte-conditioned media (ACM) followed by flow cytometry analysis for surface marker expression, cytokine production, and the induction of cytolytic mediators. Interestingly, T-cells activated in ACM, but not UCM or SCM, showed an attenuated phenotype highlighted by decreased CD44 and PD-1 expression, diminished cytokine production (IFN-γ/TNF-α) and reduced induction of cytolytic mediators (granzyme B/perforin). These observations were also true in obese, relative to lean, patients with B-ALL where we found that T-cells purified from the peripheral blood mononuclear cells (PBMCs) failed to produce significant levels of TNF-α when stimulated with PMA/Ionomycin. In all, these results demonstrate that adipocyte-secreted factors directly compromise the function of endogenous T-cells, which phenocopies T-cell defects observed in obese relative to lean pediatric patients with B-ALL. We next assessed the impact of adiposity on malignant cells by culturing human B-ALL cell lines in the conditioned mediums described above and performed flow cytometric analysis to assess their surface expression of the B-cell lineage antigen CD19 and proteins that modulate immunity. In addition to being a marker for B-cells, CD19 is the primary target of the T-cell based immunotherapies Blinatumomab and CAR T-cells directed against B-ALL cells. Surprisingly, when human B-ALL cells were co-cultured with adipocytes, every cell line tested (n=6) exhibited lower surface CD19 expression with 5 out 6 reaching statistical significance. Furthermore, adipocyte-secreted factors alone were sufficient to reduce CD19 surface levels on B-ALL cells in 2 of the 6 cell lines tested. Human B-ALL cells cultured in ACM, but not UCM or ACM, also upregulated their surface expression of the immunoinhibitory proteins HVEM, PD-L1, and PD-L2. These results demonstrate that adipocytes directly induce the downregulation of CD19 on human B-ALLs and increase their immune evasive capacity. Given these observations, we hypothesized that adipocyte-secreted factors would compromise T-cell-based immunotherapies targeting CD19-expressing B-ALL cells. To this end, primary human T-cells were engineered to express a CD19-directed chimeric antigen receptor (CAR). CAR T-cells and human B-ALL cells were separately pre-treated for 24 hours in UCM, SCM or ACM followed by co-culture for cytolytic analysis using Annexin-V/PI staining. Adipocyte-secreted factors significantly inhibited CAR T-cell mediated killing of CD19-expressing B-ALL cells at 4 hours. In addition to CAR T-cells, we tested the leukemia killing efficacy of the bispecific T-cell engager, Blinatumomab. After 3 days of culture, we found that Blinatumomab significantly increased the killing capacity of endogenous T-cells with 60-80% of B-ALL cells being killed after 3 days of culture in UCM and SCM. In contrast, we found that ACM significantly compromised the efficacy Blinatumomab with only 30% of B-ALL cells being killed over 3 days when co-cultured with human T-cells. Our pre-clinical data highlights the negative impact of an adipose-rich microenvironment on T-cell function and B-ALL immunogenicity, which subsequently compromises the efficacy of multiple classes of immunotherapies targeting CD19. Disclosures No relevant conflicts of interest to declare.

Developing a novel biologic for the treatment of high-risk B cell acute lymphoblastic leukemia

Abstract The subtype of B cell acute lymphoblastic leukemia (B-ALL) at highest risk for relapse overexpresses surface CRLF2 (CRLF2 B-ALL) and disproportionately affects Hispanic children and adults. CRLF2 partners with the IL-7Ra to form a receptor complex activated by the cytokine, Thymic Stromal Lymphopoietin (TSLP). TSLP activates the JAK/STAT and PI3/AKT/mTOR pathways which are known to promote cell survival and proliferation. Our in vitro studies of CRLF2 B-ALL cells showed that supra-physiological levels of TSLP (1 ng/ml) induced a complete shutdown of TSLP receptor signaling, following the initial activation event. This effect was dose dependent and did not occur at physiological levels (20 pg/ml) of TSLP. This signal shutdown was correlated with the loss of surface IL-7Ra. Using a novel patient-derived xenograft (PDX) model that expresses human TSLP (hTSLP, mouse TSLP does not activate the human TSLP receptor) created in our lab, we showed that high dose hTSLP virtually eliminated human CRLF2 B-ALL cells in vivo. The goal of work presented here was to test whether treatment of CRLF2 B-ALL PDX with high doses of recombinant hTSLP (rhTSLP) could re-capitulate anti-tumor effects of hTSLP observed in our engineered mice and to evaluate its effects on prolonged survival. After 24 days of treatment, rhTSLP reduced the leukemia cell burden by 90% in treated PDX as compared to control PDX that received physiological levels of rhTSLP. Similarly, survival was prolonged in PDX mice receiving high-dose rhTSLP as compared to controls: all 11 control mice died before any of the 11 treated mice died. Taken together, these data suggest that TSLP has promise as a biologic for the treatment of CRLF2 B-ALL.

Elevated Levels of Interleukin-9 in Obese Microenvironments Promote Leukemia Progression and Chemoresistance in B-Cell Acute Lymphoblastic Leukemia Cells

Background: The CDC reports that obesity rates in children and adolescents have more than tripled since the 1970's (Ogden et al., 2006; Ogden et al., 2016). Obese pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) have inferior overall survival compared to lean patients, with obese patients having a 2.5-fold increased risk of an adverse event and an almost 4-fold greater risk of death compared to non-obese patients (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012). Emerging data suggest that factors in the obese microenvironment directly promote chemoresistance in B-ALL cells (Ehsanipour et al., 2013; Sheng et al., 2016). Despite the emerging epidemiological and clinical studies highlighting the negative impact of obesity on cancer outcomes, there remains a lack of understanding of the molecular mechanisms underlying the dismal outcomes of obese patients with B-ALL. Methods: Using a global cytokine profiling array, we found that multiple cytokines were secreted at low levels in a lean microenvironment compared to the obese microenvironment. We decided to focus solely on cytokines exclusively present at high concentrations in the obese microenvironment relative to the lean microenvironment in order to determine their impact on the function of murine and human B-ALL cell lines. We also utilized the diet-induced murine model of obesity to determine how survival and treatment outcomes differ in lean and obese mice challenged with B-ALL. Furthermore, we determined how obesity altered the function of B-ALL cells in pediatric patients using primary samples obtained through the Aflac Leukemia and Lymphoma Biorepository. Results: We made the novel finding that Interleukin-9 is elevated in obese microenvironments and alters the function of human B-ALL cells. It has been previously shown that interleukin-9 (IL-9) promotes chemoresistance in diffuse large B-cell lymphoma and is associated with a worse prognosis in patients with B-cell chronic lymphocytic leukemia (Chen, Lv, Li, Lu, &amp; Wang, 2014; Lv, Feng, Ge, Lu, &amp; Wang, 2016); however, the impact of IL-9 on B-ALL development is unknown. We have found that IL-9 levels were significantly increased in adipose-rich microenvironments. Human B-ALL cells exposed to these environments upregulated the IL-9 receptor (IL-9R), which was not observed in stromal-cell rich microenvironments. Stimulating human B-ALL cells with recombinant IL-9 (rIL-9) promoted cell progression and extensive proliferation over 3 days of culture. Furthermore, rIL-9 stimulation of human B-ALL cells activated survival pathways (STAT3) which coincides with the induction of chemoresistance to methotrexate. Conclusions: We have found that IL-9 levels are elevated in obese microenvironments and alters the function of human B-ALL (increased proliferation, activation of survival pathways, and induction of chemoresistance). In ongoing murine studies, we will determine if the survival of obese mice with B-ALL is prolonged when chemotherapy treatment and IL-9 neutralizing strategies are combined. Disclosures No relevant conflicts of interest to declare.

Genetic Analysis of B-Cell Acute Lymphoblastic Leukemia Dissemination to the Central Nervous System Identifies Clonal Selection and Therapeutic Vulnerability

Without prophylactic therapy, B-cell Acute Lymphoblastic Leukemia (B-ALL) spreads to the leptomeninges of the central nervous system (CNS) in up to 70% of patients. CNS involvement is more common in certain high risk B-ALL subgroups, including patients with KMT2A (MLL)-translocations, and disease relapse in the CNS carries a poor prognosis. The genetic determinants and biology of B-ALL dissemination to the CNS are poorly defined and therefore therapies targeting the drivers of CNS disease are lacking. Whereas B-ALL exhibits significant subclonal diversity that contributes to functional heterogeneity and disease relapse, recent reports suggest similar clonal composition of bone marrow (BM) and CNS disease, with the potential for CNS dissemination being a universal property of B-ALL cells (Williams et al. 2016, Bartram et al. 2018). Furthermore, functional studies of leptomeningeal disease have focused on the invasion of B-ALL cells into the CNS but limited studies have addressed the selection of genetic clones with the ability to grow within the subarachnoid space.To better define the evolutionary history and biology of leptomeningeal B-ALL we performed targeted DNA, SNP copy number, RNA sequencing, and functional analysis on cells isolated from matched BM and CNS tissue of patient derived xenografts (PDX) generated from a cohort of paired diagnosis and relapse samples from 14 pediatric and adult B-ALL patients of varying cytogenetics. The majority of primary patient samples yielded CNS disease 20 weeks after intrafemoral injection into NSG mice. CNS disease burden was higher in PDXs derived from relapsed B-ALL samples. Human B-ALL cells isolated from the CNS of PDXs retained competence to repopulate disease in the BM, spleen, and CNS upon serial transplantation. Targeted DNA sequencing results analyzed using a Bayesian clustering method revealed different genetic clonal composition between matched BM and CNS cells in approximately half of the xenografts. PDXs from relapse samples were more likely to show genetic discordance between the BM and CNS. Copy number analysis also confirmed frequent genetic discordance between cells isolated from the BM and CNS from individual PDXs. Interestingly, in one patient all PDXs generated from the relapse sample displayed chromosome 6p and 17p hemi-deletions that were unique to the CNS. In total, PDXs from four patients showed recurrent enrichment of specific lesions in CNS-engrafting cells, suggesting that transit to and/or survival within the subarachnoid space can be the product of selection for genetic clones with increased CNS tropism.RNA-seq of matched BM and CNS cells derived from 45 of the primary PDXs demonstrated that CNS-isolated cells were transcriptionally distinct from their matched BM. These differences were most pronounced in samples from patients with MLL-AF4 translocations, whose CNS isolated cells grouped together in multi-dimensional scaling. Using GSEA, the most highly CNS-enriched gene sets in MLL samples were related to mRNA translation initiation and polypeptide elongation. Translation-related gene sets are similarly enriched in the blasts of MLL B-ALL patients with CNS disease in the COG 9906 study. CNS-isolated cells from PDXs of MLL patients exhibited altered rates of protein synthesis compared to matched BM-isolated cells. Treatment of PDXs with the clinically-approved translational inhibitor omacetaxine mepesuccinate (OMA) effectively decreased rates of translation in CNS-engrafting cells. Moreover, OMA reduced leukemia burden nearly 4-fold in PDXs bearing established CNS infiltration generated from two MLL patients.Our data represent an advance in the understanding of B-ALL CNS disease. We present a rich resource of genomic and transcriptomic data from xenografts spanning multiple B-ALL subgroups across diagnosis and relapse and have identified selection for genetically and biologically distinct clones in the CNS, contrary to the current model. Furthermore, we demonstrate that in MLL patients, dysregulation of protein synthesis occurs at CNS dissemination and targeting this process is a novel therapeutic paradigm that may benefit patients with CNS disease. DisclosuresMullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding.

Precise Modeling of IKZF1 Alterations in Human B-Cell Acute Lymphoblastic Leukemia Cell Lines Reveals Distinct Chemosensitivity, Homing, and Engraftment Properties

Around 20% of pediatric and the majority of adults with B-cell acute lymphoblastic leukemia (B-ALL) suffer relapse, and prognosis after relapse is very poor. Therefore, identifying those at risk for treatment failure and improving their outcome is imperative. In B-ALL, deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are associated with an increased risk of relapse. IKZF1 encodes the IKAROS protein, which is a master lymphoid regulatory transcription factor and chromatin remodeler. Somatic IKZF1 lesions are thought to be secondarily acquired, arising in lymphoblasts with existing driver genetic lesions, most commonly co-occurring with BCR-ABL1 fusion, activating kinase fusions of Ph-like disease and deregulated DUX4 and ERG. In B-ALL, mono- or bi-allelic deletions of the entire gene, as well as intragenic deletions occur. One of the most common perturbations of IKZF1 in B-ALL is an intragenic deletion of a 50-kilobase (kb) region containing exons 4-7, resulting in the expression of a dominant-negative isoform, IK6. Recently published clinical data show potentially conflicting results over the benefits of therapy intensification in IKZF1-mutant cases (Clappier et al., 2015; Hinze et al., 2017; & Yeoh, et al., 2018). Human cell models of these deletions are needed, as there may be unknown functional differences among mutation types, and the available body of data relies on clinical statistical associations, in vitro RNA interference, viral overexpression of IK6, and mouse models.We used the CRISPR/Cas9 system in the human B-ALL cell lines Nalm-6 and REH by electroporation with sgRNA-Cas9 ribonucleoprotein complexes (RNPs) to generate IKZF1-mutant clones. We identified single cell-derived clonal lines with IKZF1 frameshift mutations in one or both alleles by Sanger sequencing and TIDE decomposition. We confirmed ablation of protein expression by immunoblotting. We treated the IKZF1-mutant clonal cell lines with chemotherapeutic agents commonly used to treat B-ALL and calculated the IC50 by Annexin V/7-AAD double-negative population after 48-72 hour treatment. Compared to IKZF1-wild type Nalm-6 cells, Nalm-6 IKZF1-/- clones exhibited profound resistance to dexamethasone and modest but significant resistance to most other chemotherapeutics tested including vincristine, asparaginase, and daunorubicin. In contrast, these cell lines were more sensitive to the nucleoside analog, cytarabine (Panel A). We next analyzed gene expression profiles by RNA-seq and observed that IKZF1-/- clones are characterized by a stem cell-like gene expression signature and activation of the JAK/STAT pathway (Panel B). Transplantation into immunodeficient NOD scid gamma (NSG) mice demonstrated that IKZF1 deletion leads to enhanced engraftment, significantly increased bone marrow homing, and reduced survival time (Panel D).We also employed a novel CRISPR/Cas9 homology-directed repair (HDR) strategy to generate clonal cell lines expressing IK6 under control of the endogenous promoter, which represents a significant advantage to many previous studies utilizing viral overexpression. We electroporated the cells with sgRNA-Cas9 RNPs along with a 3kb commercially synthesized double-stranded DNA HDR template that knocks-in exon 8 with a GFP tag directly following exon 3. Using this strategy, we were able to isolate heterozygous clones (IKZF1IK6/+) from both Nalm-6 (Panel C) and REH cell lines using flow cytometry sorting for GFP-positive cells. We confirmed precise HDR by Sanger sequencing and immunoblotting. When transplanted into immunodeficient mice, IKZF1IK6/+ cells showed delayed engraftment and disease onset, but profound splenic infiltration, consistent with a more indolent, infiltrative disease phenotype (Panels D & E). Ongoing drug treatment assays suggest the chemosensitivity profiles of IKZF1IK6/+ and IKZF1IK6/- clonal cell lines are distinct from their isogenic IKZF1-/- counterparts.Our data support clinical studies reporting that IKZF1-mutated B-ALL is an aggressive, infiltrative, and treatment-resistant disease. Notable differences in drug response and in vivo dynamics in xenografts exist between IKZF1-/- cells and IKZF1IK6/+ cells. Detailed delineation of the exact IKZF1 status in ALL patients at diagnosis may be informative in more accurately determining risk stratification and the most effective therapeutic regimen. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2537: Regulation of LMO2 oncogene expression in high-risk B-cell acute lymphoblastic leukemia

Abstract LIM domain only protein 2 (LMO2) is an oncogene that is overexpressed in a subset of B-cell acute lymphoblastic leukemia (B-ALL). The mechanisms that regulate LMO2 expression in B-ALL are still unknown. Here, we show that transcription of LMO2 in B-ALL is regulated by Ikaros, a tumor suppressor protein, which is encoded by the IKZF1 gene, and whose deletion is associated with development of high-risk B-ALL. The use of global chromatin immunoprecipitation coupled with the next-generation sequencing (ChIP-seq) studies in primary human B-ALL cells and in cell lines revealed a strong Ikaros occupancy at the promoter of the LMO2 gene. Ikaros binding to the LMO2 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP). We tested the role of Ikaros in regulating LMO2 transcription in B-ALL using gain-of-function and loss-of-function experiments. A luciferase reporter assay with the LMO2 promoter showed that Ikaros directly represses LMO2 transcription. Overexpression of Ikaros in B-ALL via retroviral transduction was associated with strongly reduced expression of LMO2. Furthermore, Ikaros knock-down with shRNA, resulted in increased transcription of LMO2 in B-ALL cells. These results suggest that Ikaros represses transcription of LMO2 in B-ALL. Since Ikaros' function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 inhibition affects Ikaros-mediated repression of LMO2. Both molecular inhibition with shRNA, and pharmacological inhibition of CK2 with a specific CK2 inhibitor, CX-4945, resulted in reduced expression of LMO2. Inhibition of CK2 was also associated with increased Ikaros occupancy at the LMO2 promoter. In high-risk B-ALL that have deletion of a single copy of the IKZF1 gene, Ikaros does not bind to the LMO2 promoter. Treatment of primary B-ALL cells that have IKZF1 haploinsufficiency restores Ikaros binding to the LMO2 promoter and results in reduced LMO2 expression. In conclusion, our data demonstrate that Ikaros and CK2 regulate transcription and overall expression of the LMO2 oncogene in B-ALL. Our results identify a novel mechanism of therapeutic action of CK2 inhibitors in high-risk B-ALL—repression of LMO2 expression via restoration of Ikaros' tumor suppressor function. Citation Format: Yali Ding, Jonathon L. Payne, Shriya Kane, Elanora Dovat, Mario Soliman, Chunhua Song, Sinisa Dovat. Regulation of LMO2 oncogene expression in high-risk B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2537.

MTOR inhibition enhances efficacy of dasatinib in ABL-rearranged Ph-like B-ALL.

High-risk subtypes of B-cell acute lymphoblastic leukemia (B-ALL) include Philadelphia chromosome-positive (Ph+) B-ALL driven by the BCR-ABL1 oncogene and a more recently identified subtype known as BCR-ABL-like or Ph-like B-ALL. A hallmark of both Ph+ and Ph-like B-ALL is constitutive activation of tyrosine kinase signaling that is potentially targetable with tyrosine kinase inhibitors (TKIs). B-ALL cells also receive extracellular signals from the microenvironment that can maintain proliferation and survival following treatment with TKIs. Therefore, there is strong rationale for combining TKIs with other therapies targeting signal transduction pathways. Here we show that combinations of the ABL-directed TKI dasatinib with mTOR kinase inhibitors (TOR-KIs) are more effective than TKI alone against patient-derived Ph-like B-ALL cells harboring rearrangements of ABL1 or ABL2. We also report the establishment of a new human Ph-like B-ALL cell line that is stromal cell-independent in vitro and can be used for xenograft experiments in vivo. These findings provide rationale for clinical testing of TKI plus TOR-KIs in children and adults with Ph-like B-ALL and a new experimental tool to test promising therapeutic strategies in this poor prognosis subtype of B-ALL.