Co-Targeting of the Thymic Stromal Lymphopoietin Receptor to Decrease Immunotherapeutic Resistance in <i>CRLF2</i>-Rearranged Ph-like and Down Syndrome ALL

Background: CRLF2 (cytokine receptor-like factor 2) rearrangements occur in 50% of Philadelphia chromosome-like (Ph-like) and Down syndrome (DS)-associated B-acute lymphoblastic leukemia (ALL), resulting in aberrant cell surface overexpression of the thymic stromal lymphopoietin receptor (TSLPR) protein and constitutive intracellular JAK/STAT and other kinase pathway signaling. Children and adolescents/young adults (AYAs) with Ph-like ALL have high rates of chemoresistance and poor event-free survival and have been enriched in early-phase relapsed leukemia clinical trials testing novel therapeutics. Patients with DS-ALL also have inferior clinical outcomes and experience high rates of toxicity with conventional chemotherapy. The potential improved efficacy of addition of the JAK1/2 inhibitor ruxolitinib to chemotherapy in children and AYAs with Ph-like ALL is under active clinical trial investigation, but has not been studied to date in patients with DS-ALL, and trial results are not yet known. Chimeric antigen receptor (CAR) T cell immunotherapies targeting CD19 or CD22 have achieved impressive initial remission induction in &amp;gt;80% of children and AYAs with relapsed/refractory B-ALL, but approximately 50% of CD19CART-treated patients subsequently relapse, including a high proportion with CD19 antigen loss. As an alternative approach, we previously developed and demonstrated robust activity of TSLPRCART immunotherapy in CRLF2/TSLPR-overexpressing ALL preclinical models (Qin Blood 2015). Methods: In the current studies, we tested the hypothesis that co-targeting extracellular TSLPR with CART immunotherapy and intracellular JAK/STAT signaling with ruxolitinib would induce synergistic anti-leukemia activity in CRLF2-rearranged (CRLF2-R) Ph-like and DS-ALL using cell lines and patient-derived xenograft (PDX) models. Results: TSLPRCART monotherapy showed effective and durable inhibition of leukemia proliferation in vitro in CRLF2-R Ph-like ALL cell lines and in vivo in both CRLF2-R Ph-like ALL (n=4) and DS-ALL (n=3) PDX models. However, we unexpectedly observed fatal TSLPRCART-induced toxicity in ALL PDX mice treated at higher CART doses that correlated with high levels of in vivo inflammatory cytokine production. Interestingly, simultaneous TSLPRCART and ruxolitinib co-administration prevented early animal death, but also significantly reduced T cell proliferation, blunted cytokine production, and/or facilitated leukemia relapse. Importantly, delaying ruxolitinib co-exposure by two weeks completely preserved desired anti-leukemia TSLPRCART activity and prevented cytokine release syndrome (CRS)-type mortality, resulting in long-term ‘cure’ and survival of Ph-like ALL PDX mice. We further observed in additional Ph-like ALL PDX model experiments that ruxolitinib withdrawal following TSLPRCART-induced leukemia remission resulted in recovery of CAR T cell functionality with in vivo re-expansion of TSLPRCART, IFN-γ production, and leukemia clearance after subsequent CRLF2/TSLPR+ ALL ‘relapse’ rechallenge. These results were fully recapitulated in DS-ALL PDX models, validating JAK/STAT pathway hyperactivation and the TSLPR antigen as a critical immunotherapeutic target, and with CD19CART in orthogonal in vitro ALL cell line and in vivo PDX model studies. Conclusion: Ruxolitinib-induced inhibition of TSLPRCART and CD19CART fitness and anti-ALL activity is robust, but reversible. Biologically-appropriate time sequencing of CART immunotherapy and ruxolitinib achieved both effective acute CRS mitigation and robust long-term anti-ALL activity in preclinical models, which has high translational potential as a relapse prevention ‘maintenance’ strategy for patients with CRLF2-R Ph-like ALL or DS-ALL.