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.