Session: 631. CML: Biology and Pathophysiology, excluding Therapy: Mechanisms of Resistance and Progression in CML
Hematology Disease Topics & Pathways:
Diseases, CML, Biological Processes, Myeloid Malignancies, hematopoiesis, pathogenesis
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.
Disclosures: Marcucci: Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Merck: Other: Research Support (Investigation Initiated Clinical Trial).
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