RUNX1 loss renders hematopoietic and leukemic cells dependent on IL-3 and sensitive to JAK inhibition
Amy C. Fan, … , Purvesh Khatri, Ravindra Majeti
Amy C. Fan, … , Purvesh Khatri, Ravindra Majeti
Published August 15, 2023
Citation Information: J Clin Invest. 2023;133(19):e167053. https://doi.org/10.1172/JCI167053.
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Research Article Hematology Inflammation

RUNX1 loss renders hematopoietic and leukemic cells dependent on IL-3 and sensitive to JAK inhibition

Abstract

Disease-initiating mutations in the transcription factor RUNX1 occur as germline and somatic events that cause leukemias with particularly poor prognosis. However, the role of RUNX1 in leukemogenesis is not fully understood, and effective therapies for RUNX1-mutant leukemias remain elusive. Here, we used primary patient samples and a RUNX1-KO model in primary human hematopoietic cells to investigate how RUNX1 loss contributes to leukemic progression and to identify targetable vulnerabilities. Surprisingly, we found that RUNX1 loss decreased proliferative capacity and stem cell function. However, RUNX1-deficient cells selectively upregulated the IL-3 receptor. Exposure to IL-3, but not other JAK/STAT cytokines, rescued RUNX1-KO proliferative and competitive defects. Further, we demonstrated that RUNX1 loss repressed JAK/STAT signaling and rendered RUNX1-deficient cells sensitive to JAK inhibitors. Our study identifies a dependency of RUNX1-mutant leukemias on IL-3/JAK/STAT signaling, which may enable targeting of these aggressive blood cancers with existing agents.

Authors

Amy C. Fan, Yusuke Nakauchi, Lawrence Bai, Armon Azizi, Kevin A. Nuno, Feifei Zhao, Thomas Köhnke, Daiki Karigane, David Cruz-Hernandez, Andreas Reinisch, Purvesh Khatri, Ravindra Majeti

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Figure 1

Targeting the endogenous RUNX1 locus in human HSPCs using CRISPR and homology directed repair.

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Targeting the endogenous RUNX1 locus in human HSPCs using CRISPR and hom...
(A) Umbilical CB is enriched for CD34 using magnetic activated cell sorting (MACS) and expanded in serum-free media with SCF, TPO, FLT3L, IL-6, and UM-171. After 2 days, cells are nucleofected with RUNX1- or AAVS1- targeting sgRNA-Cas9 ribonucleoprotein and exposed to AAV6 carrying donor DNA for 8 hours. Three days after editing, CD34+mCherry+GFP+ cells are sorted and plated for in vitro and in vivo assays or molecular profiling. (B) Recombinant AAV6 vector carries arms of homology flanking fluorescent reporter (FP) transgenes encoding GFP or mCherry as donor DNA for HDR at DNA double-stranded breaks generated by RUNX1-targeting sgRNA-Cas9. LHA, left homology arm; RHA, right homology arm; pA, poly-A. (C) Left: Biallelic modified GFP+mCherry+ double-positive cells are not present in controls lacking sgRNA-Cas9. mC, mCherry. Right: Quantification of double-positive HDR editing efficiency at the AAVS1 safe harbor locus and RUNX1 locus in CD34+ HSPCs. n = 61 CB donors. (D) Left: Schematic of in/out PCR spanning target-donor junction to confirm integration of the RUNX1-KO vector in the endogenous RUNX1 locus. Right: Agarose gel showing in/out PCR product in RUNX1-KO CD34+ HSPCs. (E) qPCR detection of fold change in RUNX1 expression relative to AAVS1 control and normalized to HPRT1. Unpaired t test: *** P < 0.001. n = 2 CB donors, 3 replicates. (F) Western blot of RUNX1 protein in unmodified and RUNX1-KO CD34+ HSPCs.