Genomic and functional analysis of leukemic transformation of myeloproliferative neoplasms

Abstract

Patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard AML therapies. We performed genomic profiling of post-MPN AML samples; these studies demonstrate somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells. Consistent with these data, expression of JAK2V617F combined with Tp53 loss led to fully penetrant AML in vivo. JAK2V617F-mutant, Tp53-deficient AML was characterized by an expanded megakaryocyte erythroid progenitor population that was able to propagate the disease in secondary recipients. In vitro studies revealed that post-MPN AML cells were sensitive to decitabine, the JAK1/2 inhibitor ruxolitinib, or the heat shock protein 90 inhibitor 8-(6-iodobenzo[d][1.3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purine-6-amine (PU-H71). Treatment with ruxolitinib or PU-H71 improved survival of mice engrafted with JAK2V617F-mutant, Tp53-deficient AML, demonstrating therapeutic efficacy for these targeted therapies and providing a rationale for testing these therapies in post-MPN AML.

Keywords: cancer biology; genetics; leukemia; myeloproliferative neoplasm; targeted therapy.

Fig. 1.

Genetic events in leukemic transformation of chronic-phase MPNs. (A) Frequency of mutations in post-MPN AML samples (n = 33). Hash marks indicate structural rearrangements (for scale reference, alterations were observed at a frequency of 3.1% beginning with ARID1A and extending to ZRSR2 in this representation). (B) Circos representation of co-occurring mutations in JAK2-mutant post-MPN AML. (C) Circos representation of co-occurring mutations in JAK2-wildtype post-MPN AML. (D) VAF of the most frequently mutated genes in post-MPN AML, in paired chronic-phase MPN and AML. (E) Representative analysis of VAF of mutations and bone marrow blasts occurring at chronic MPN phase and AML stage from a single patient.

Fig. 2.

JAK2V617F collaborates with Tp53 loss to induce AML. (A) Bone marrow from Tp53-null (Tp53-KO) or wild-type C57BL/6 mice was harvested and transduced with either JAK2V617F- or MigR1-containing retrovirus. Transduced cells then were injected into lethally irradiated congenic recipients. (B) Survival of mice injected with Tp53-KO bone marrow transduced with JAK2V617F (Tp53-KO/JAK2V617F) was reduced significantly compared with control arms (P < 0.01, t test). (C–E) Trend of WBC (C), HCT (D), and PLT (E) in Tp53-KO/JAK2V617F mice compared with control arms from day 14 posttransplantation to day 100. (F) WBC count of recipients injected with Tp53-KO/JAK2V617F bone marrow was significantly greater than that of p53WT/JAK2V617F control (P < 0.05, t test) at day 100 posttransplantation, with a trend toward increase compared with other control arms. Blood counts displayed are derived from two independent experiments. (G) Representative spleens from killed animals. (H and I) Spleen (H) and liver (I) weights demonstrating increased organ weights in Tp53WT/JAK2V617F mice. (J and K) Representative peripheral blood smear (J) and bone marrow cytospin (K) from Tp53WT/JAK2V617F mice demonstrate increased numbers of intermediate to large blasts with round and irregular nuclei, high nuclear:cytoplasmic ratios, and finely stippled chromatin, consistent with acute leukemia; maturing myeloid and erythroid precursors are markedly reduced (Wright–Giemsa stain). (L and M) Representative spleen sections (L) and bone marrow (M) from Tp53WT/JAK2V617F mice demonstrating increased blasts consistent with acute leukemia (H&E stain).

Fig. 3.

Tp53-KO/JAK2V617F mice are characterized by an expansion of hematopoietic stem and erythroid progenitor populations. (A) Immunophenotypic analysis demonstrating an increase in the frequency of LSK cells compared with control mice. (Upper) Representative FACS plots. (Lower Left) Quantification of bone marrow and spleen LSK populations. (Lower Right) Histogram of c-kit expression on live, nucleated cells in spleen. (B) The CD71/Ter119 double-positive population and the CD71 single-positive population are increased significantly in bone marrow of Tp53-KO/JAK2V617F mice (P < 0.05, t test). (C) Analysis of spleen cells demonstrates an increase in the MEP population in Tp53-KO/JAK2V617F mice compared with controls. Three mice per group were analyzed.

Fig. 4.

Loss of Tp53 in the JAK2V617F setting confers increased self-renewal to committed progenitor populations. (A) Methylcellulose replating assay demonstrating enhanced self-renewal of Tp53-KO/JAK2V617F whole bone marrow. (B) Serial transplantation of whole spleen cells derived from leukemic Tp53-KO/JAK2V617F mice demonstrates significantly reduced latency with serial transplantation (P < 0.05 primary versus secondary, P < 0.05 secondary versus tertiary, log-rank test). (C) Transplantation of spleen cells from Tp53-KO/JAK2V617F leukemic mice results in the development of leukemia in both lethally and sublethally irradiated recipient mice. (D) Transplantation of the MEP population from Tp53-KO/JAK2V617F mice with leukemic phenotype into lethally irradiated recipients results in the development of leukemia in mice surviving longer than 20 d (n = 7).

Fig. 5.

Pharmacologic inhibition of JAK2 as well as degradation of JAK2 alone and in combination with other therapies inhibits Tp53-KO/JAK2V617F leukemia. (A–E) The clonogenic capacity of Tp53-KO/JAK2V617F leukemic bone marrow cells in methylcellulose is inhibited on exposure to the JAK1/2 inhibitor ruxolitinib (A), the JAK1/2 inhibitor CTY387 (B), the Hsp90 inhibitor PU-H71 (C), decitabine (D), and a combination of decitabine and ruxolitinib (INCB18424) (E). For each condition, 40,000 spleen-derived cells were plated. (F) Clonogenic capacity of TP53 mutant/JAK2V617F human leukemic bone marrow cells in methylcellulose is inhibited on exposure to either ruxolitinib or PU-H71. (G) Treatment with either ruxolitinib or PU-H71 significantly prolongs survival (P < 0.01, log-rank test) of Tp53-KO/JAK2V617F leukemic mice relative to vehicle, and treatment with PU-H71 significantly prolongs survival compared with ruxolitinib (P < 0.01, log-rank test). PU-H71 was discontinued 1 wk after all ruxolitinib-treated mice were deceased. (H) Bone marrow cytospins demonstrating a predominance of blasts in vehicle-treated mice (Left), compared with increasing evidence of granulocytic maturation in mice treated with ruxolitinib (Center) and PU-H71 (Right). (I and J) Treatment with ruxolitinib or PU-H71 results in significant reductions in spleen (I; P < 0.05, P < 0.01, respectively) and liver (J; P < 0.01 for both treatments relative to vehicle) weights. PU-H71 significantly reduced spleen weight compared with ruxolitinib as well (P < 0.01, t test). (K) Treatment with ruxolitinib results in expansion of CD3⁺ cells (P < 0.05, t test), and treatment with PU-H71 results in expansion of CD3⁺ (P < 0.05, t test), CD11b⁺⁺ (P < 0.01, t test), and B220⁺ (P < 0.01, t test) populations in spleens of treated mice. (L) The proportion of CD71⁺ cells is reduced in bone marrow of ruxolitinib- and PU-H71–treated mice (P < 0.05, t test, for PU-H71) and in spleen of ruxolitinib- and PU-H71–treated mice (P < 0.05, t test, for PU-H71). (M) Treatment with ruxolitinib or PU-H71 results in the expansion of CD11b⁺ (P < 0.05 for ruxolitinib, and P < 0.01 for PU-H71, t test) and B220⁺ (P < 0.01 for PU-H71, t test) cells in bone marrow of treated mice. (N) Western blot demonstrating reduction in phospho-JAK2 and phospho-STAT5 levels in mice treated with ruxolitinib or PU-H71 relative to placebo. **P < 0.05.