CRLF2/Tslpr Overexpressing Acute Lymphoblastic Leukemia Relapse Is Driven By Chemotherapy-Induced TSLP from Bone Marrow Stromal Cells

Despite 5-year survival rates nearing 90%, leukemia is the most frequent cause of death from cancer in children with treatment failure primarily caused by relapse after remission. Therefore, it is critical that we identify new therapies that specifically address leukemic persistence during therapy. A cohort of acute lymphoblastic leukemia (ALL) patients that have an increased risk of relapse and consequently poorer overall survival have leukemia that overexpresses cytokine receptor-like factor 2/thymic stromal lymphopoietin receptor (CRLF2/TSLPR). CRLF2/TSLPR overexpressing patients have rates of relapse nearly double the rate of non-overexpressing patients despite comparable remission rates to low-risk patients and account for half of the cases of high risk Philadelphia-like ALL. We hypothesize that TSLP/TSLPR axis may promote relapse of ALL through overexpressed TSLPR, which sensitizes leukemic blasts to low levels of TSLP in bone marrow (BM) niches promoting survival. To test this theory we generated a TSLPR overexpressing syngeneic murine leukemia (TSLPRhigh) through viral transduction of a transplantable pre-B cell ALL line (TSLPRlow). This TSLPRhigh leukemia has comparable TSLPR expression levels to what is found on human ALL that overexpress TSLPR and the transduced TSLPR is functional with increased phosphorylation of STAT5 protein in response to TSLP stimulation. When ALL lines were injected into immunocompetent mice, we observed an 8 fold difference in the percentage of TSLPRhigh vs. TSLPRlow ALL in the BM 5 days after injection, when leukemia accounted for less than 5% of the BM corresponding to an early stage of ALL progression. Interestingly, in vitro and in vivo cell growth and late-stage lethality were no different between the TSLPRhigh and TLSPRlow ALL indicating that TSLP does not alter ALL proliferation. From this data we can infer that TSLP/TSLPR signaling is likely most critical at early stages of leukemia development when BM stromal niches are intact. Hypothesizing that these niches are the source of TSLP ligand we sought to identify the cells responsible for secreting TSLP in the BM.  Indeed, basal levels of TSLP mRNA are present in the BM, but the expression is quite low. Interestingly, TSLP mRNA and protein were markedly induced in murine BM stromal cell lines and primary BMSCs by the inflammatory cytokines IL-1a and TNF-a as has previously been reported for other types of TSLP-producing human cells. Since it is well established that cytotoxic chemotherapeutics can mediate an inflammatory response in patients, we investigated whether cytotoxic agents can cause release of inflammatory cytokines from BM cell populations. We treated primary murine BMSCs, BM hematopoietic cells, and ALL lines with low doses of chemotherapy and observed that the pyrimidine analog cytarabine (Ara-C) was particularly potent in upregulating IL-1a expression from BM hematopoietic cells and ALL and not in BMSCs suggesting that inflammatory cytokine release by hematopoietic cells may induce TSLP production by BMSCs. We set out to determine if IL-1a administration to mice could enhance the early progression of ALL in vivo. Indeed, we found that TSLP mRNA and protein were elevated in the BM and serum of mice injected with IL-1a respectively and that there was significant increase in the percentage of early ALL infiltration in the BM of mice bearing TSLPRhigh ALL but not TSLPRlow ALL suggesting a dependence on high levels of expression of TSLPR to respond to IL-1a -induced TSLP production. In addition, we confirmed the necessity of TSLPR-driven early ALL progression on the presence of the TSLP ligand as we observed no significant increase in TSLPRhigh ALL in TSLP deficient mice after stimulation with IL-1a. Furthermore, the early increase in TSLPRhigh leukemia burden can be reversed by using a TSLP blocking antibody demonstrating that targeting the TSLP/TSLPR axis may be therapeutically relevant. These data demonstrate that TSLP secreted in the BM induced by inflammatory cytokines such as IL-1a can drive accelerated early progression of ALL. These inflammatory cytokines can be induced by cytotoxic agents suggesting that chemotherapy can indirectly provide an unintended advantage to TSLPR overexpressing ALL. Finally, we postulate that therapies targeting the TSLP signaling axis would decrease the risk of relapse in TSLPRhigh ALL particularly in the context of standard therapy.