Inhibition of Inflammatory Signaling in Tet2 Mutant Preleukemic Cells Mitigates Stress-Induced Abnormalities and Clonal Hematopoiesis

Abstract

Inflammation is a risk factor for cancer development. Individuals with preleukemic TET2 mutations manifest clonal hematopoiesis and are at a higher risk of developing leukemia. How inflammatory signals influence the survival of preleukemic hematopoietic stem and progenitor cells (HSPCs) is unclear. We show a rapid increase in the frequency and absolute number of Tet2-KO mature myeloid cells and HSPCs in response to inflammatory stress, which results in enhanced production of inflammatory cytokines, including interleukin-6 (IL-6), and resistance to apoptosis. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis, resulting in increased expression of a novel anti-apoptotic long non-coding RNA (lncRNAs), Morrbid, in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss with regard to hyperactivation of Stat3 and Morrbid. In vivo, pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2 mutant mice rescues inflammatory-stress-induced abnormalities in HSPCs and mature myeloid cells, including clonal hematopoiesis.

Keywords: Morrbid; Tet2; inflammation; preleukemic; stem cells.

Figure 1.. Tet2-KO mice exhibit transient but amplified neutrophil and HSPCs in response to acute inflammatory challenge

(A) Hematologic changes in the peripheral blood (PB) of LPS-treated wildtype and Tet2-KO mice over a 7-day period. A single dose of LPS (i.p., 0.8 mg/kg) was administered to adult wildtype or Tet2-KO mice (3~4 month old). n=4–10 mice per group, results are pooled from multiple experiments. (B) LPS induces aberrant emergency granulopoiesis in Tet2-KO mice, revealed by flow cytometry analysis using neutrophil markers Mac1 and Ly6G. n=4 mice per group. (C) A schematic demonstrating hematopoietic hierarchy. (D) Representative flow cytometry profiles showing gating strategy and changes in various bone marrow HSPC subsets. (E) Quantification of the frequency (Freq.) and absolute cell number (No.) of CMP, LSK and HSCs on day 0 and day 2 post LPS treatment in adult wildtype and Tet2-KO mice. n =4 mice per group. Results are representative of two independent experiments. P value: * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant. Statistical analysis performed by unpaired, two-tailed Student’s t-test. See also Figure S1 for additional supporting data.

Figure 2.. LPS-stressed Tet2-KO bone marrow cells maintain repopulating advantage

(A) A schematic describing primary and secondary competitive bone marrow transplantation (cBMT) assay. The age of the mice for donor cells was about 3~4 months old. (B-C) Tet2-KO bone marrow cells with or without LPS treatment manifest significantly higher engraftment in primary and secondary recipients compared to wildtype controls. (D-F) Identical number of LSK cells were purified from wildtype and Tet2-KO mice pre-and post-LPS treatment and subjected to ex vivo CFU assay (E) and in vivo cBMT assay (F), respectively. Transplant experiments were conducted as described in (D). Data in (B) are from a representative experiment (n=5 recipients for cBMT analysis, mean ± s.e.m.). Results are representative of two independent experiments. Data in (C) are from a single experiment (n=5 recipients per group, mean ± s.e.m.). Data in (E) is from pooled analysis of two mice per group performed in replicates of 4 (mean ± s.e.m, n=4 replicates). P value: * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant. Statistical analysis performed by unpaired, two-tailed Student’s t-test. See also Figure S2 for additional supporting data.

Figure 3.. Tet2-KO hematopoietic progenitor cells show sustained cell survival and heightened activation of Morrbid in response to acute inflammatory challenge

(A-B) Level of apoptosis in Lin- cells and LSK cells pre and post LPS treatment as assessed by Annexin-V/7-AAD staining. A, representative flow profile of Annexin/7-AAD staining. B, quantification of apoptosis level. (C) Differential expression of pro-apoptotic and pro-survival genes in Lin- cells pre- and post-LPS treatment. (D) A scheme for two possible mechanisms by which Tet2 loss induces hyperactivation of Morrbid: one is through direct regulation of Morrbid promoter and another one is through cytokine-modulated phosphorylation of Stat3 (pStat3), which may bind to Morrbid promoter and activate its expression. (E-F) Tet2-KO HSPCs maintain elevated expression of pStat3, revealed by flow cytometry (E) and by western blot (F). (G) Stat3 binds to Morrbid locus revealed by CHIP-qPCR enrichment assay. Shown is relative enrichment in binding (1% of input as unit 1). Data in (A) is a representative profile of flow cytometry. Data in (B-G) are from a representative experiment (n=4 mice per group, mean ± s.e.m.). Results are representative of two independent experiments. P value: * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant. Statistical analysis performed by unpaired, two-tailed Student’s t-test. See also Figure S3 for additional supporting data.

Figure 4.. Tet2-KO mice show increased expression of IL-6 in serum and in various bone marrow subsets

(A) Multiple cytokines/chemokines were increased on day 1 and/or day 2 post- LPS treatment in Tet2-KO mice, compared to wildtype controls. (B-C) Intracellular flow cytometry analysis (ICFC) of IL-6 expression in total bone marrow cells and in Lin- bone marrow cells pre- and post-LPS treatment. (D) Expression of IL-6 in indicated bone marrow subsets as assessed by flow cytometry and MFI quantification. (E-F) QRT-PCR analysis of Il6 and Ccl2 expression in bone marrow Lin- cells derived from adult and juvenile pre- and post-LPS treated wildtype and Tet2-KO mice. Data in (A) are from a single experiment (n=4 mice per group, mean ± s.e.m.). Data in (B) and (C) are from a representative experiment. Results are representative of two independent experiments. Data in (D), (E) and (F) are from a representative experiment (n= 4 mice per group, mean ± s.e.m.). Results are representative of two independent experiments. P value: * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant. Statistical analysis performed by unpaired, two-tailed Student’s t-test. See also Figure S4 for additional supporting data.

Figure 5.. IL-6 stimulates a Shp2/Stat3/Morrbid-mediated pro-survival pathway in vitro, which is inhibited by pharmacologic inhibitors SHP099 and E3330.

(A) A schematic describing an abbreviated form of canonical TLR4/NFκB/IL-6 and putative IL-6/Stat3/Morrbid signaling pathway. (B) IL-6 promotes the survival of Lin- cells in liquid culture assay. (C) IL-6 induces the expression of activation of Morrbid in wildtype and Tet2-KO Lin- cells. (D) Lin- cells expressing Shp2E76K, a gain-of-function isoform of Shp2, manifest decreased apoptosis, decreased expression of Bim and elevated expression of Morrbid, compared to wildtype controls. (E and F) E3330 or SHP099 treatment of Tet2-KO Lin-negative bone marrow cells corrects the aberrant colony-forming ability of Tet2-KO cells in primary and secondary replating assay. E3330, 0.5 μM. SHP099, 0.1 μM. (G) E3330 or SHP099 treatment of Tet2-KO Lin-negative bone marrow cells inhibits IL-6 induced cell survival. (H) E3330 or SHP099 inhibits Stat3 binding to the Morrbid promoter as revealed by CHIP-qPCR assay. E3330, 0.5 μM. SHP099, 0.1 μM. Data in (B-H) are from a representative experiment (n=3 to 4 per group, mean ± s.e.m.). Experiments were repeated twice. P value: * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant. Statistical analysis performed by unpaired, two-tailed Student’s t-test. See also Figure S5 for additional supporting data.

Figure 6.. E3330 and SHP099 treatment represses clonal expansion of Tet2-KO HSPCs and prevents aberrant early signs of MPN in both young and aged Tet2 mutant mice

(A) A schematic showing the experimental procedure used for generating chimeric mice mimicking clonal expansion of Tet2-KO cells. Tet2-KO bone marrow cells (CD45.2) were mixed with BoyJ bone marrow cells (CD45.1) at a ratio 1 to 5 (100K: 500K). Two months post-transplant, the chimeric mice were subjected to continuous treatment with LPS (0.8 mg/kg, i.p. injection every other day for 1 month) or a daily injection of E3330 or SHP099 (oral gavage, 20 mg/kg for E3330, 50 mg/kg for SHP099 for 3 months). Chimerism in peripheral blood was analyzed monthly at indicated time points. (B) Clonal expansion of Tet2-KO HSPCs is heightened by continuous administration of LPS. (C) Clonal expansion of Tet2-KO HSPCs is repressed by daily injection of E3330 or SHP099. (D) Long-term treatment of juvenile Tet2-KO mice with E3330 or SHP099 results in the rescue of multiple phenotypic defects associated with Tet2 deficiency including frequency of CMPs, LSKs, HSCs, expression of Morrbid, Bim, IL-6 and pStat3 as well as spleen weight. (E) Short-term treatment of aged Tet2-KO mice with E3330 or SHP099 prevents early hematological signs of CMML in peripheral blood. E3330, 20 mg/kg and SHP099, 50 mg/kg, oral gavage. Transplant data in (B) and (C) are derived from using 5 recipient mice per group, mean ± s.e.m. 15 doses of LPS were given at 0.8 mg/kg over a period of one month (i.p. injection, every other day). E3330 (20 mg/kg) and SHP099 (50 mg/kg) were administered by oral gavage daily for 12 weeks in (D) or for 2 weeks in (E). Drug treatment studies in (D) and (E) are pooled data from two independent experiments (n=4 to 5 mice per group, mean ± s.e.m.). P value: * P < 0.05, ** P < 0.01. n.s., not significant. Statistical analysis performed by unpaired, two-tailed Student’s t-test. See also Figure S6 for additional supporting data.

Figure 7:. Genetic loss of Morrbid represses the growth advantage in Tet2-deficient preleukemic cells

(A) A schematic showing the experimental procedures for testing genetic requirement of Morrbid in Tet2 deficiency-mediated aberrant hematopoietic phenotypes in naïve mice or LPS-challenged mice, and in ex vivo repopulation assays and in vivo cBMT assays. (B) Tet2^+/−; Morrbid^+/− mice fail to manifest amplified “emergency hematopoiesis” seen in Tet2^+/− mice upon LPS treatment. (C) Rescue of splenomegaly in Tet2^+/−;Morrbid^+/− mice relative to Tet2^+/− mice. (D) Tet2^+/− HSPCs with Morrbid haplo-insufficiency corrects the enhanced self-renewing ability of Tet2^+/− HSPCs in an in vitro serial replating assay. (E) Tet2^+/− HSPCs with Morrbid haplo-insufficiency rescues the enhanced clonal expansion seen with Tet2^+/− HSPCs as assessed in a chimeric cBMT assay. Data in (B), (C), and (E) are from a representative experiment (n = 4 to 5 mice per group, mean ± s.e.m.). Data in (D) is pooled from two mice performed in replicates of 4. Data in (E) are from 4 to 5 recipient mice for every group at every time point [4 to 16 weeks] (n=4 to 5, mean ± s.e.m.). Similar results were seen in another independent experiment (n=4 to 5 mice per group; analyzed at 8 weeks). P value: * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., not significant. Statistical analysis performed by unpaired, two-tailed Student’s t-test. See also Figure S7 for additional supporting data.