Microrna-142 Deficiency Promotes Chronic Myeloid Leukemia (CML) Transformation from Chronic Phase (CP) to Blast Crisis (BC)

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm resulting from the BCR-ABL1 fusion gene that encodes a constitutively activated tyrosine kinase (TK). Although TK inhibitors (TKIs) induce disease remission and prolonged survival in CML patients, a subset are resistant and progress from chronic phase (CP) to blast crisis (BC) with poor prognosis. Understanding the molecular mechanisms of transformation from CP to BC is necessary in the development of effective treatments.

Here, we used the inducible SCLtTA/BCR-ABL transgenic CP CML model to study the molecular mechanism of disease evolution. Upon tetracycline withdrawal to induce BCR-ABL expression, both the SCLtTA/BCR-ABL homozygous (homo, i.e., SCLtTA^+/+BCR-ABL^+/+, hereafter called BCR-ABL) and heterozygous (het, i.e., SCLtTA^+/-BCR-ABL^+/-) mice developed and died of CP CML without developing BC CML, implying that BCR-ABL dosage is insufficient to induce transformation.

MicroRNA (miR)-142 is highly expressed in hematopoietic cells with a critical role in normal hematopoiesis. In miR-142 knockout (KO)(miR-142^−/−) mice, hematopoietic stem and progenitor cells expanded with a decrease of hematopoietic output. Loss of miR-142 function has been reported in lymphoma, acute lymphocytic leukemia and acute myeloid leukemia. Of note, we also observed lower levels of miR-142 in CD34⁺CD38^- cells from patients with BC CML versus (vs) patients with CP CML. Thus, we hypothesized that miR-142 insufficiency may promote CML transformation from CP to BC.

To test our hypothesis, we generated miR-142 KO BCR-ABL (i.e., miR-142^−/−BCR-ABL) mice and observed increasing leukemic blasts over time after BCR-ABL induction in the blood and bone marrow (BM), but not in miR-142 wt (miR-142^+/+)BCR-ABL controls even when the latter became moribund. MiR-142^−/−BCR-ABL mice had larger spleens and significantly shorter survival [median: 26 vs 54 days (d); p<0.0001] than miR-142^+/+BCR-ABL controls. Of note, while both homo (miR-142^−/−) and het (miR-142^+/−) miR-142 KO BCR-ABL mice eventually developed BC CML, the former had a significantly faster progression to BC and shorter survival (median: 26 vs 45 d; p=0.003) than the latter, suggesting miR-142 deficiency alone is sufficient to initiate BC transformation in the CP CML model in a dose-dependent manner. Importantly, all these features were recapitulated in congenic recipient mice transplanted with BM Lin^-Sca-1⁺c-Kit⁺ cells (LSKs, 2000/mouse) from diseased miR-142^−/−BCR-ABL mice, suggesting LSKs were enriched in leukemic stem cells and able to reproduce BC. Of note, in an RNA-seq analysis comparing LSKs from diseased miR-142^−/−BCR-ABL (BC) and miR-142^+/+ BCR-ABL(CP) mice, 504 genes were found differentially expressed. Gene set enrichment analysis (GSEA) showed only four pathways differentially expressed (upregulated); three [i.e., oxidative phosphorylation, glycolysis and adipogenesis] regulating cell metabolism and the fourth regulating protein secretion.

Next, we developed a novel CpG-miR-142 mimic oligonucleotide, hereafter called CpG-M-miR-142, to restore miR-142 levels. Treatment with CpG-M-miR-142 (20mg/kg/day, iv, 4 weeks) on day 2 after BCR-ABL induction significantly prolonged survival of miR-142^−/−BCR-ABL mice compared with CpG-scramble (SCR) (75% vs 33% survival rate at day 40 after BCR-ABL induction; median survival: not reached vs 25 d; p=0.03).

Since we observed lower miR-142 levels in TKI-resistant vs TKI-sensitive CML patients (p=0.02), we selected LSKs from diseased miR-142^−/−BCR-ABL and miR-142^+/+BCR-ABL mice and exposed them to TKI nilotinib (NIL; 2µM) or vehicle for 72 hours to evaluate if downregulation of miR-142 was associated with TKI resistance. We observed lower apoptosis and higher cell growth in NIL-treated miR-142^−/−BCR-ABL LSKs vs NIL-treated miR-142^+/+BCR-ABL LSKs. The decreased sensitivity of miR-142^−/−BCR-ABL LSKs to TKI was rescued by treatment with CpG-M-miR-142. CpG-M-miR-142 (2µM) plus NIL significantly increased apoptosis and reduced cell growth in miR-142^−/−BCR-ABL LSKs compared with SCR+ NIL.

We showed a key role of miR-142 deficiency in the transformation of CP CML to BC CML associated with deregulation of metabolic pathways. Restoring miR-142 expression in vivo with CpG-M-miR-142 significantly decreased the BC transformation rate, prolonged survival of miR-142^−/−BCR-ABL mice and may increase sensitivity to TKIs.