Interferon-Activated Gene Signatures Associate with Chemotherapy Resistance and Sensitivity to JAK-Inhibition in Refractory ETP-ALL
Authors: Xu Jason , Sussman Jonathan , Yang Austin , Yoshimura Satoshi , Chen Changya , Elghawy Omar , Vincent Tiffaney , Diorio Caroline , Bandyopadyay Shovik , Hu Jianzhong , Li Alexander Lai , Chen Gregory M. , Chen Chia-Hui , Shraim Rawan , Newman Haley , Ding Yang , Li Elizabeth , Yu Wenbao , Thadi Anusha , Ahn Kyung Jin , Pölönen Petri , Elsayed Abdelrahman , Frank David , Raetz Elizabeth A. , Loh Mignon L. , Wood Brent L. , Winter Stuart S. , Dunsmore Kimberly P. , Devidas Meenakshi , Qu Chunxu , Pounds Stanley , Hunger Stephen P. , Mullighan Charles G. , Yang Jun J. , Tan Kai , Teachey David T. 
Published: December 2024

Introduction
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid malignancy in which optimal therapeutic approaches for relapsed/refractory disease and biomarkers for upfront risk stratification are currently unknown.We previously reported integrated scRNA and scATAC-seq analysis on 40 T-ALL cases that identified a BCL2-inhibitor sensitive bone-marrow progenitor-like (BMP-like) tumor sub-population that is highly resistant to conventional therapy and found across all immunophenotypic subtypes of T-ALL. Further analysis of blasts from BMP-high and BMP-low T-ALL patients revealed one outlier patient (PASKMG) within ETP-ALL cases that had refractory disease (Day 29 bone marrow MRD &gt;20%) despite a low proportion of BMP-like blasts. Notably, this patient was the only patient that harbored a BMP-low signature (&lt;10% blasts in BMP-like state) and T-lineage-high signature (&gt;50% blasts in pro-T/pre-T state) with poor response to conventional therapy. We hypothesized that transcriptomic and epigenetic characterization of PASKMG would elucidate an additional novel mechanism of treatment failure.
Results
Differential expression analysis comparing PASKMG leukemic blasts to other ETP blasts from patients with induction failure (n=5, all BMP-high) revealed a strong upregulation of 35 interferon-alpha related gene products, including 19 genes within the STAT1-driven IFN-related DNA damage resistance signature (IRDS) previously identified in chemotherapy/radiation refractory solid tumors. Intersection of scRNA and scATAC data revealed STAT1, STAT5B, STAT3, IRF7/9 to be top differentially expressed transcription factors (TFs) with differentially accessible TFs motifs, consistent with STAT activation seen in IRDS expressing carcinomas. Interestingly, our analysis also identified a GATA2+, CD117+, CD33+, CD63+ MEP-like subpopulation with high expression of IRDS genes. This leukemia subpopulation was retained in PDX models of PASKMG and harbored a strong inflammatory signature with enrichment of neutrophil degranulation and cell adhesion pathways.
We next hypothesized that tumor-intrinsic IRDS expression could associate with a unique leukemic micro-environment. Indeed, across non-leukemic cell subsets, we detected expression of antiviral gene expression associated with interferon signaling, including upregulation of HLA components and PLCG2 signaling. Compared to 21 other single-cell profiled ETP-ALL BM aspirates, PASKMG harbored a uniquely inflammatory microenvironment dominated by NK and effector T-cell subsets, rather than by non-activated Naïve-T cells.
Prior studies have indicated unique sensitivity of IRDS+ solid tumors to JAK/STAT inhibition. While JAK-inhibition has been explored as a targeted therapy for T-ALL, initial results indicate highly heterogenous responses amongst patients. To test if IRDS could be a biomarker of JAK inhibitor sensitivity in refractory ETP-ALL, we expanded blasts from PASKMG and other ETP-ALL patients in patient derived xenograft models. Single-cell transcriptomic analysis of PDX expanded blasts indicated strong retention of IRDS signatures unique to PASKMG. Within in-vitro drug screening and in-vivo experiments, we found that PASKMG had markedly increased sensitivity to multiple JAK inhibitors compared to other ETP-ALL and T-ALL.
Discussion
Here, we report the identification of an IRDS resistance signature in leukemia that is associated with targetable JAK/STAT signaling, inflammatory microenvironment, mast-cell-like tumor subpopulation, and increased sensitivity to JAK inhibition. To our knowledge, this is the first report of IFN-related DNA damage resistance signature in liquid tumors; in contrast, in solid tumors, IRDS positivity is known to be a marker of chemo and radiation resistance. Our PDX data indicating unique vulnerability of IRDS+ ETP-ALL to JAK-inhibitors are translationally promising for successful implantation of JAK/STAT inhibitors in T-ALL. We propose further study of interferon activation in T-ALL and other leukemias for prospective identification of patients that may benefit from JAK inhibitor targeted therapy.
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*JX, JS, and AY contributed equally to this work
**DT and KT co-supervised this work