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, & Wang, 2014; Lv, Feng, Ge, Lu, & 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