KDM6B protects T-ALL cells from NOTCH1-induced oncogenic stress

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors. While activating NOTCH1 mutations are the dominant genetic drivers of T-ALL, epigenetic dysfunction plays a central role in the pathology of T-ALL and can provide alternative mechanisms to oncogenesis in lieu of or in combination with genetic mutations. The histone demethylase enzyme KDM6A (UTX) is also recurrently mutated in T-ALL patients and functions as a tumor suppressor. However, its gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed, suggesting it may be necessary for sustaining the disease. Here, we used mouse and human T-ALL models to show that KDM6B is required for T-ALL development and maintenance. Using NOTCH1 gain-of-function retroviral models, mouse cells genetically deficient for Kdm6b were unable to propagate T-ALL. Inactivating KDM6B in human T-ALL patient cells by CRISPR/Cas9 showed KDM6B-targeted cells were significantly outcompeted over time. The dependence of T-ALL cells on KDM6B was proportional to the oncogenic strength of NOTCH1 mutation, with KDM6B required to prevent stress-induced apoptosis from strong NOTCH1 signaling. These studies identify a crucial role for KDM6B in sustaining NOTCH1-driven T-ALL and implicate KDM6B as a novel therapeutic target in these patients.

Figure 1:. Kdm6b is essential for maintenance of NOTCH1-mutant T-ALL cells

A) T-ALL (CD45.2+ GFP+) engraftment in peripheral blood of mice transplanted with Control (n=37), Kdm6b-HET (n=28), and Kdm6b-KO (n=20) NICD-expressing cells four-weeks post-transplant. B) Kaplan-Meier plot of mice transplanted with Control (n=37), Kdm6b-HET (n=28), and Kdm6b-KO (n=20) NICD-expressing T-ALL cells. C) T-ALL burden in the bone marrow of moribund mice. Black shapes indicate mice that did not develop disease during the experimental timecourse. D) T-ALL disease burden in the peripheral blood of individual mice over the experimental timecourse. E) Representative spleens from mice transplanted with T-ALL cells of indicated genotypes. F) Spleen weights from moribund mice. G) T-ALL burden in the spleens of moribund mice. Black shapes indicate mice that did not develop disease during the experimental timecourse. **** p<0.0001. Data analyzed by log-rank Mantel-Cox test (B), or one-way ANOVA with Tukey correction for multiple comparisons (A, C, F, G).

Figure 2:. T-ALL cells undergo apoptosis in the absence of Kdm6b

A) Hierarchical clustering of genes with >2-fold expression change (p-value <0.05, FDR <0.05) between control and Kdm6b-KO T-ALL cells. B) Gene set enrichment analysis (GSEA) identified three significantly enriched pathways involved in cell cycle regulation downregulated in Kdm6b-KO T-ALL cells. C) Representative flow cytometry plot showing Ki67 cell cycle analysis of T-ALL cells from the peripheral blood of mice. D) Cell cycle analysis of control and Kdm6b-KO T-ALL cells from the blood of recipient mice four-weeks post-transplant. E) Representative flow cytometry plots showing AnnexinV apoptosis analysis of T-ALL cells from the peripheral blood of mice. F) Quantification of apoptotic NICD-expressing T-ALL cells from blood of recipient mice four-weeks post-transplant. * p<0.05, **** p<0.0001. Data analyzed by two-tailed t-test (D, F).

Figure 3:. Pro-tumorigenic function of Kdmb6 in T-ALL is demethylase-dependent

A) T-ALL (CD45.2+ GFP+) engraftment in peripheral blood of mice transplanted with Control (n=14), Kdm6b-HET (n=15), Kdm6b-KO (n=15) and Kdm6b-CD (n=15) NICD-expressing cells eight-weeks post-transplant. B) Kaplan-Meier plot of mice transplanted with Control (n=15), Kdm6b-HET (n=15), Kdm6b-KO (n=15) and Kdm6b-CD (n=15) T-ALL cells. **** p<0.0001. Data analyzed by one-way ANOVA with Tukey correction for multiple comparisons (A) or log-rank Mantel-Cox test (B).

Figure 4:. KDM6B is required to sustain disease in a subset of T-ALL patients

A) Patient-derived xenograft of a representative KDM6B “non-responder” adult T-ALL patient sample showing peripheral blood engraftment of human T-ALL cells (hCD45+ hCD7+), overall survival, bone marrow engraftment of T-ALL cells from moribund mice and variant allele frequency of CRISPR edited targets. B) Patient-derived xenograft of a representative KDM6B “responder” adult T-ALL patient sample showing peripheral blood engraftment of human T-ALL cells (hCD45+ hCD7+), overall survival, bone marrow engraftment of T-ALL cells from moribund mice and variant allele frequency of CRISPR edited targets. C) Dynamic analysis of CRISPR edits in adult T-ALL cells over the experimental timecourse showing VAF of AAVS1 control and KDM6B segregated into responders versus non-responders. VAF is normalized to the initial CRIPSR efficiency in cells prior to transplant. For KDM6B VAF, values represent compiled normalized values for two independent gRNAs per sample.

Figure 5:. KDM6B restrains expression of pro-apoptotic genes in KDM6B-dependent T-ALLs

A) Cell viability of KDM6B responder (orange) and non-responder (grey) patient samples after 48-hours in vitro treatment with increasing concentrations of GSK-J4. B) Cell viability of T-ALL patient samples following CRISPR editing with indicated gRNAs after 48-hours in vitro treatment with increasing concentrations of GSK-J4. C) VAF of indicated CRISPR edits in primary T-ALL patient cells following seven-days in vitro culture. D) Unsupervised clustering of normalized RNA-seq gene expression data of primary T-ALL patient samples targeted for either AAVS1 or KDM6B by CRISPR gene editing. E) Gene set enrichment of KDM6B responder versus non-responder samples targeted with AAVS1 gRNA (control conditions). Numbers atop each bar show number of genes within specified geneset. F) Gene set enrichment of KDM6B responder versus non-responder samples targeted with KDM6B gRNAs. Numbers atop each bar show number of genes within specified geneset. G) Heatmap of gene expression in HALLMARK “Apoptosis” gene signature that was significantly enriched in responder T-ALL patient samples after targeting with KDM6B gRNAs. H) Representative flow cytometry plots showing AnnexinV apoptosis staining of T-ALL patient sample after CRISPR targeting with indicated gRNAs and seven-days in vitro culture. I) Relative fraction of apoptotic cells (normalized to average AAVS1 AnnexinV+ cells per patient) within T-ALL patient samples after CRISPR targeting with indicated gRNAs and seven-days in vitro culture. Orange and blue values for KDM6B group indicate two different gRNAs. ** p<0.01. Data analyzed by two-tailed t-test relative to the AAVS1 control for each individual patient (I).

Figure 6:. Genetic signature of KDM6B dependence in T-ALL

A) Normalized mRNA expression of KDM6B and KDM6A in KDM6B responder versus non-responder T-ALL patient samples. B) Variant allele frequency of NOTCH1 mutations in KDM6B responder versus non-responder T-ALL patient samples. C) Distribution of mutations across NOTCH1 in KDM6B responder versus non-responder T-ALL patient samples. D) Co-occurrence of genes recurrently mutated in T-ALL in KDM6B responder versus non-responder T-ALL patient samples. Patients with multiple mutations in NOTCH1 are denoted by multiple boxes within NOTCH1 column. E) Distribution of mutations across NOTCH1 in KDM6B responder patient samples with multiple NOTCH1 mutations.

Figure 7:. KDM6B protects T-ALL cells from strong NOTCH1 signaling-induced apoptosis

A) Western blot showing protein levels of cleaved NOTCH1 (NICD) in KDM6B responder versus non-responder T-ALL patient samples. B) Western blot showing protein levels of cleaved NOTCH1 (NICD) in KDM6B responder versus non-responder T-ALL patient samples 48-hours post-targeting with indicated gRNAs. C) RNA-seq expression levels of classic NOTCH1 signaling genes and NOTCH1 T-ALL target genes in human KDM6B responder versus non-responder T-AL patient samples, and control and Kdm6b-KO mouse T-ALL cells. D) Western blot comparing protein levels of cleaved NOTCH1 (NICD) in cells transduced with indicated NOTCH1 mutant retroviral constructs compared to the empty vector control. E) Quantification of apoptotic NOTCH1^L1601P-ΔPEST-expressing T-ALL cells from blood of recipient mice four-weeks post-transplant. F) Kaplan-Meier plot of mice transplanted with Control (n=15), Kdm6b-HET (n=13), and Kdm6b-KO (n=13) NOTCH1^L1601P-ΔPEST-expressing T-ALL cells. * p<0.05, ** p<0.01. Data analyzed by two-tailed t-test (C).