Danielle Wolfe Cohen, MD, on Mechanisms of Relapse in B-Cell Acute Lymphoblastic Leukemia

I am here today at the American Society of Hematology Annual Meeting and Exposition, and I'm delighted to share my research with you. Although outcomes for children with acute lymphoblastic leukemia have improved dramatically, survival for the 10 to 20% of children who relapse remains dismal. Our laboratory focuses on discovering genetic and epigenetic alterations leading to relapse and discovered a novel super enhancer adjacent to the S100A8 and S100A9 genes shared by a number of patients at relapse, implicating a role in driving resistance. S100A8 and S100A9 are members of the S100 Calcium Binding Family and have been shown to mediate intrinsic drug resistance in cancer. However, our previous work using engineered cell lines showed that S100A8 and A9 alone did not influence drug growth, drug sensitivity, or clonal potential. We reasoned that co-regulate genes might be driving the clonal advantage. Single-cell sequencing data was performed on four diagnosis and relapse pairs, one of which had increased S100A8 and A9 at relapse, and this suggested that the IL6 receptor was upregulated along with S100A8 and A9 at relapse, and this revealed that the IL6 pathway was the IL6 JAK STAT three signaling pathway. Our hypothesis is that S100A8 and A9 and a co-expressing blast interact with the tumor immune microenvironment via the IL6 IL6 receptor axis to create an inflammatory response that ultimately leads to this resistance that we see to validate co-expression of S100A8 and A9 and the IL6 receptor at relapse with a much larger cohort. We calculated a Pearson correlation coefficient using our previously published bulk microarray expression data on 49 patient pairs and demonstrated a significant positive correlation between S100A8 and A9 and IL6 receptor expression. We further supported the co-regulation of the IL6 receptor and S100A8 and A9 in an unbiased way using RNA sequencing data from 176 paired samples from children enrolled on the Children's Oncology Group study ALL-1331. A total of 671 genes were found significantly co-regulated with both S100A8 and A9 in acute myeloid leukemia and this included toll-like receptor four and I gamma amongst others to determine the impact of the IL6 receptor expression on drug resistance, we generated a panel of isogenic L cell lines expressing an empty vector or S100A8 and A9 with and without the IL6 receptor. While we did not observe significant differences in chemo sensitivity in the S100A8 and A9 overexpressing cell lines, we did find that co-expression with the IL6 receptor provided a clonal advantage to cell lines under glucocorticoid exposure specifically, and this is a hallmark of disease relapse in B-ALL. The IL6 receptor-mediated resistance was only observed when cells were exposed to IL6 either by culturing along with HS five condition media or recombinant human IL6. We confirmed activation of downstream signaling as indicated by phosphorylation of STAT three only in the lines expressing the IL six receptor. After exposure to the IL 6 and STAT three activation was inhibited by pretreatment with either tocilizumab, which is an IL six receptor antagonist or ruxolitinib, which is a JAK one and two inhibitor. Importantly, pretreatment with tocilizumab or ruxolitinib led to resensitization to glucocorticoids in the IL six receptor expressing cell lines. Overall, our data suggests that tumor escape is mediated by the acquisition of an inflammatory phenotype that includes increased IL six receptor expression on blasts activated by IL six, secretion from the surrounding tumor immune microenvironment that ultimately leads to resistance. The clinical significance of our work is amplified by the impact of S100, A eight and A nine and the IL six receptor on event-free survival. Patients with expression above the median have inferior event-free survival, so created S 100, A eight and A nine. Proteins are known to remodel the tumor immune microenvironment, and we are currently testing whether this remodeling also leads to a protective niche in B-ALL. Inhibition of stat signaling in this subgroup of relapsed patients may be a promising avenue to pursue in the future.