Histone demethylase KDM4C is a functional dependency in JAK2-mutated neoplasms

Abstract

Mutations of the JAK2 gene are frequent aberrations in the aging hematopoietic system and in myeloid neoplasms. While JAK-inhibitors efficiently reduce hyperinflammation induced by the constitutively active mutated JAK2 kinase, the malignant clone and abundance of mutated cells remains rather unaffected. Here, we sought to assess for genetic vulnerabilities of JAK2-mutated clones. We identified lysine-specific demethylase KDM4C as a selective genetic dependency that persists upon JAK-inhibitor treatment. Genetic inactivation of KDM4C in human and murine JAK2-mutated cells resulted in loss of cell competition and reduced proliferation. These findings led to reduced disease penetrance and improved survival in xenograft models of human JAK2-mutated cells. KDM4C deleted cells showed alterations in target histone residue methylation and target gene expression, resulting in induction of cellular senescence. In summary, these data establish KDM4C as a specific dependency and therapeutic target in JAK2-mutated cells that is essential for oncogenic signaling and prevents induction of senescence.

Fig. 1. KDM4C is a specific dependency in JAK2-mutated cells.

A Scatter plot showing the in vitro depletion scores of genes in JAK2-mutated cells versus non-JAK2-mutated cell lines according to the DepMap Avana CRISPR-Cas9 (Q4/21) database. Data points represent the median value of each sgRNA set. Scores of genes with dependencies (indicated by dependency scores < −0.5) in JAK2-mutated cells but no dependency in non-JAK2-mutated cells (dependency score > −0.5) are highlighted in red. B Venn diagram showing the number of JAK2-specific in vitro hits and the number of JAK2-V617F specific target genes as determined by phospho-proteome analyses [10]. Overlapping genes with high expression in JAK2-mutated cell lines are indicated in red. C Expression and functional dependency of KDM4C in JAK2-mutated versus non-JAK2-mutated cell lines. D Dependency scores of all KDM-gene-family members in myeloid cancer cell lines. Dependencies (dependency score < −0.5) in JAK2-mutated cell lines are indicated in blue, total number of dependencies in black.

Fig. 2. KDM4C is highly expressed in primary MPN patient cells.

A Quantification of KDM4C mRNA expression in peripheral blood granulocytes derived from patients diagnosed with myeloproliferative neoplasms (MPN; n = 129) compared to healthy donor controls (n = 12). B KDM4C mRNA expression across different MPN subtypes (PV = 24, ET = 29, MF = 31, MPN-U = 6) and healthy donor controls (n = 12).

Fig. 3. KDM4C is a JAK-inhibitor persistent functional vulnerability.

A Schematic depicting experimental setup of the in vitro CRISPR-Cas9 screen in JAK2-mutated HEL cells. Pharmacologic treatment was performed for 12 days with 200 nM ruxolitinib (RUX) versus DMSO control. B Dot-plot of ranked differential CRISPR-Cas9 screening hits between the RUX-treated and control condition. The Y axis shows the gene-rank, on the X axis the differential beta scores (Δbeta-scores) are plotted. RUX-“persistent” dependencies are highlighted in red.

Fig. 4. Inactivation of KDM4C results in loss of cell competition and reduced proliferation.

A Schematic depicting negative-selection competition assay. B Results of the negative-selection competition assay showing the relative percentage of RFP + sgRNA+ cells over time after transduction of JAK2-mutated HEL cells with indicated sgRNAs targeting luciferase control (sg-Luc), KDM4C (sg2-KDM4C; sg3-KDM4C) or RPA3 (positive control, sg-RPA3). n > 3. C Proliferation assayed by cell counting after trypan blue exclusion for Ba/F3-V617F-Cas9-Blast cells, SET-2-Cas9-Blast cells and HEL-Cas9-Blast cells transduced with sgRNAs targeting KDM4C, RPA3, or a non-targeting control (sgLuc). n > 3 independent experiments. D Schematic representation of the experimental procedure for CRISPR-Cas9 mediated knockout of KDM4C in human HEL cells followed by transplantation into xenograft mice at two different dilutions (30,000 cells; 60,000 cells). E Violin plot showing spleen weight of recipient animals at the timepoint of analysis (n = 9; two-tailed t-test). F Kaplan–Meier survival curves of humanized NXG recipient mice injected with two different dilutions of CRISPR-Cas9 edited human HEL cells.

Fig. 5. Loss of KDM4C leads to differential methylation of target histones and induction of cellular senescence.

A Western blot analysis in BaF3/JAK2-V617F_Cas9_Blast, SET-2_Cas9_Blast and HEL_Cas9_Blast cells following CRISPR-Cas9 knockout using KDM4C specific sgRNAs (KDM4C-sg2, -sg3) or non-targeting control (LUC). B Immunofluorescence analysis of H3K36me3 and Hoechst-staining in SET-2_Cas9_Blast and HEL_Cas9_Blast cells following CRISPR-Cas9 knockout using KDM4C specific sgRNAs (KDM4C-sg2, -sg3) or non-targeting control (LUC). C GSEA showing enrichment of genes related to senescence and autophagy in cancer. Plotted are normalized enrichment scores (NES). D Heat map of differentially expressed genes in KDM4C deleted HEL cells versus non-targeting control (LUC); n = 4. Red zones represent higher gene expression (upregulation), blue zones represent lower gene expression (downregulation). E Representative cytospins of SA-beta-Gal-staining of KDM4C depleted HEL cells compared to non-targeting control (sgLUC). Cells exposed to daunorubicin (Dauno) serve as positive control for SA-beta-Gal staining. F Bar graph depicting quantification of SA-bet-Gal staining as depicted in E. n = 3 independent replicates, two-tailed t-test.