Complementary and countervailing actions of Jak2 and Ikk2 in hematopoiesis in mice

Hyperactivation of JAK2 kinase is a unifying feature of human Ph- myeloproliferative neoplasms (MPNs), most commonly due to the JAK2 V617F mutation. Mice harboring a homologous mutation in the Jak2 locus exhibit a phenotype resembling polycythemia vera. NFκB pathway hyperactivation is present in myeloid neoplasms, including MPNs, despite scarcity of mutations in NFκB pathway genes. To determine the impact of NFκB pathway hyperactivation in conjunction with Jak2 V617F, we utilized Ikk2 (Ikk2-CA) mice. Pan-hematopoietic Ikk2-CA alone produced depletion of hematopoietic stem cells and B cells. When combined with the Jak2 V617F mutation, Ikk2-CA rescued the polycythemia vera phenotype of Jak2 V617F. Likewise, Jak2 V617F ameliorated defects in hematopoiesis produced by Ikk2-CA. Single-cell RNA sequencing of hematopoietic stem and progenitor cells revealed multiple genes antagonistically regulated by Jak2 and Ikk2, including subsets whose expression was altered by Jak2 V617F and/or Ikk2-CA but partly or fully rectified in the double mutant. We hypothesize that Jak2 promotes hematopoietic stem cell population self-renewal, whereas Ikk2 promotes myeloid lineage differentiation, and biases cell fates at several branch points in hematopoiesis. Jak2 and Ikk2 both regulate multiple genes affecting myeloid maturation and cell death. Therefore, the presence of dual Jak2 and NFκB hyperactivation may present neomorphic therapeutic vulnerabilities in myeloid neoplasms. Floxed mice containing the alleles Ikk2-CA (R26StopFLikk2ca) and Jak2 V617F (Mullally et al., 2010 Cancer Cell) were crossed to Vav1-iCre (B6.Cg-Tg(Vav1-cre)A2Kio/J), and cis-NF-κBEGFP (FVB.Cg-Tg(HIV-EGFP,luc)8Tsb/J) from Jackson Laboratory. This generated hematopoietic specific expression of the Ikk2-CA (Ikk2 S177E, S181E) and Jak2 V617F alleles, together with an NF-κB responsive EGFP reporter. Mice harboring these alleles were crossed to generate double conditional mutant mice, which were then crossed with Vav1-iCre to generate hematopoietic specific double mutant mice. Mice harboring the Vav1-iCre allele but lacking any other mutant alleles were used as controls in this study. LSK (Lin-Sca-1+Kit+) stem and progenitor cells were sorted from pooled bone marrow derived from five mice of each of four genotypes: Vav1-iCre control, Ikk2-CA, Jak2 V617F, and double mutant. Bone marrow cells from 5 mice of each genotype were pooled, and Kit+ cells were purified using Cd117 microbead kit on AutoMACS separator (Miltenyi). Kit+ cells were stained with an antibody panel (Fisher et al, 2023 Experimental Hematology) Bone marrow cells from 5 mice of each genotype were pooled, and Kit+ cells were purified using Cd117 microbead kit on AutoMACS separator (Miltenyi). Kit+ cells were stained with antibody panel and sorted on MoFlo sorter (Beckman Coulter). Sorted cells were resuspended at 1000/μL density in 1x PBS with 0.04% BSA. Cells were subjected to droplet bead capture, followed by cell lysis, reverse transcription, and amplification according to TotalSeqA protocol from 10X Genomics (Pleasanton, CA). Single cell derived cDNA libraries were sequenced on NovaSeq S4 cell sequencer (Illumina, San Diego, CA). CellRanger (10x Genomics, version 3.0.1) was used for transcript alignment, counting, and inter-library normalization of single cell samples. Aggr function was used to merge genotype libraries into a single dataset, and initial visualization utilized LoupeBrowser version 6.0 (10x Genomics). Cluster and gene expression analysis utilized Seurat version 4.0.0. Cells with <500 or >7500 genes captured per cell were removed from analysis, as were cells with 10% or more mitochondrial transcripts. RNA reads were normalized with LogNormalize and data were scaled. Unsupervised cell clustering was performed using an unanchored version of the uniform manifold approximation and projection (UMAP) algorithm in Seurat. Differential gene expression analysis between populations/samples of interest was performed utilizing FindAllMarkers with Wilcoxon rank sum test. Gene set enrichment analysis of DEGs was run using fgsea and the Hallmark gene set from msigdbr. Cell populations were identified from cluster expression of canonical HSC and progenitor population markers and NFκB target genes.