Bcor Mutations Cooperate with Tet2 Deficiency to Promote RAS Transformation of Mouse Hematopoietic Progenitors
Authors: Fares Iman , Vedula Rahul S. , Vahanvaty Shabbir M. , Waters Christopher S , Luskin Marlise R. , Kumari Priti , McConkey Marie , Charles Anne , Copson Kevin , Abel Gregory A. , Stone Richard M. , Ebert Benjamin L. , Lindsley R. Coleman 
Published: October 2019

Somatic mutations can have highly stereotyped positions in the myeloid clonal hierarchy and distinct patterns of co-occurring mutations. Gene mutations that cause aberrant activation of RAS/MAPK signaling are typically late events in myeloid disease progression and are closely associated with leukemic transformation. We hypothesized that the phenotypic output of oncogenic RAS signaling is dynamically reprogrammed during leukemogenesis based on evolving genetic and epigenetic context.

To identify genetic alterations that may modulate RAS-mediated transformation, we evaluated 1273 adults with myelodysplastic syndrome, including 150 with mutations in NRAS, KRAS, PTPN11, CBL, RIT1, NF1, or FLT3. Somatic mutations in ASXL1 (q<0.0001), RUNX1 (q<0.0001), EZH2 (q<0.0001), BCOR (q=0.0002), and STAG2 (q=0.001) were most significantly associated with co-occurring RAS pathway mutations, compared to those without RAS pathway mutations, while TP53 mutations were less frequent (q=0.059). We validated these observations in an independent cohort of 6343 unselected patients, including 1081 patients harboring either RAS pathway mutations (n=651),TP53 mutations (n=494), or both (n=57).

To define the effects of sequential acquisition of driver mutations, we developed a mouse serial transplantation model of somatic myeloid transformation. First, we used in vivo pI:pC treatment to induce biallelic inactivation of Tet2 in adult Mx1-Cre/Tet2flox/floxmice. After 12 weeks, we purified Tet2-/-or control hematopoietic stem and progenitor cells (HSPCs) and used CRISPR/Cas9 to separately introduce inactivating mutations in Ezh2, Asxl1-exon12, Stag2, or Bcor, then evaluated their functional effects using ex vivo serial replating or in vivo competitive transplantation. Tet2-/-HSPCs with control sgRNA showed a modest enhancement of serial replating compared to Tet2-wild type HSPCs, while Tet2-/-HSPCs Asxl1, Stag2, and Bcor, but not Ezh2 sgRNA had markedly enhanced serial replating capacity (>6 platings in all replicates). In primary transplantation, secondary mutations caused in vivo clonal advantage after 16 weeks, but never resulted in histologic transformation to acute leukemia.

We next evaluated the impact of tertiary NRASG12Dmutations in each pairwise Tet2-/-CRISPR combination (Asxl1, Bcor, Ezh2, Stag2, control). We purified HSPCs from recipient mice 16 weeks after primary transplantation, transduced with a lentiviral NRASG12Dexpression vector and transplanted into secondary recipients. Recipients of Tet2/Bcor/NRAS, Tet2/Asxl1/NRAS, or Tet2/Ezh2/NRAS cells succumbed to CD11b+myeloid disease with variable latency in Bcor (14 days), Ezh2 (50 days), and Asxl1 (120 days) cells, suggesting that combined Tet2 and PRC1/2 alterations may modify the effects of oncogenic RAS signaling.

To determine whether pre-existing epigenetic mutations cooperate to alter the transcriptional response to acute oncogenic stress compared to wild type cells, weperformed RNA-seq 12 and 24 hours after induced expression of NRASG12D in isogenic immortalized mouse progenitor cells deficient for Tet2, Bcor, or both Tet2 and Bcor. We observed rapid activation of inflammatory and cellular senescence programs in all conditions, suggesting a genotype-independent immediate early response to oncogenic signaling. However, we also identified genotype-specific regulation of tumor suppressor and cell cycle checkpoint pathways. While Cdnk1a expression was strongly induced in all conditions, Cdnk2a expression (and p16Ink4a and p19ARF protein levels) was preferentially upregulated in the context of Bcor deficiency. Moreover, expression of the p53 negative regulator Mdm2 was increased 11-fold in Tet2/Bcor-deficient cells, but only 4 to 5-fold in wild type, Tet2-, or Bcor-deficient cells. Tet2/Bcor-deficient cells were significantly more sensitive to treatment with the Mdm2 antogonist, Nutlin, upon induction of NRAS expression than were wild-type cells, suggesting that Mdm2 overexpression directly mediates acquired tolerance of oncogene stress.

These human genetic data and mouse models suggest that epigenetic alterations occurring during early myeloid leukemogenesis may enable evasion of oncogene protection mechanism. Bcor mutations can pair with initiating Tet2 mutations to facilitate RAS mediated transformation while incurring a dependency on Mdm2 overexpression.

Disclosures

No relevant conflicts of interest to declare.