Downregulation of Mir-142 Promotes Leukemia Growth in Philadelphia Chromosome-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL): A Possible Novel Therapeutic Target?
Authors: Wang Huafeng , Zhang Bin , Wang Wei-Le , Zhao Dandan , Li Ling , Wu Herman , Nguyen Le Xuan Truong , Meng Weixu , McDonald Tinisha , Pichiorri Flavia , Kuo Ya-Huei , Chen Jianjun , Carlesso Nadia , Aldoss Ibrahim , Pullarkat Vinod A. , Al Malki Monzr , Stein Anthony S. , Boldin Mark , Jin Jie , Marcucci Guido 
Published: October 2019

Abstract
The Philadelphia (Ph) chromosome or t(9;22) results in the generation of a fusion gene, namely BCR/ABL1, which encodes a chimeric protein with aberrant tyrosine kinase activity that drives leukemia cell growth and survival. This molecular/cytogenetic aberration occurs in ~20%-30% of ALL cases and confers poor prognosis. Ph+ ALL patients (pts) are often referred for allogeneic hematopoietic stem cell transplantation (alloHCT), although more recently BCR-ABL-specific tyrosine-kinase inhibitors (TKIs) and immunotherapeutic approaches seemingly induced long-term remission in some patients. Nevertheless, it is still a challenge to determine which Ph+ ALL of the pts could be treated more conservatively without alloHCT. Thus identification of new prognostic biomarkers and/or therapeutic targets may be helpful. Regulation of short non-coding microRNAs(miRNAs) associated with initiation and progression of acute leukemia has been reported. miR-142(both miR-142-3p and miR-142-5p) is expressed at a relatively high level in hematopoietic tissue, and plays a role in myeloid lineage differentiation. In fact, low miR-142-3p expression was associated with myeloid differentiation failure, and miR-142 mutations was reported to promote acute myeloid leukemia (AML). More recently, Kramer et al demonstrated a role of miR-142 in lymphopoiesis by showing that miR-142 deficiency impaired B cell production in a miR-142 knock-out(ko) mouse model (Blood. 2015).
Here, we first investigated if miR-142 levels were altered in ALL pts. Analysis of a publically available miRNA expression dataset(GSE23024) showed lower level of miR-142-3p, but not miR-142-5p in Ph+ ALL pts(n=10) vs. healthy donors(n=7;p=0.0093); while no significant differences were observed in Ph- pre-B ALL pts(n=61) vs. healthy donors (n=7). In ALL Tg(P190-BCR/ABL) transgenic mice(Ph+ ALL; Nature. 1990), we found bone marrow (BM) miR-142-3p level to be ~2.3-fold lower than those in the wild-type (wt) controls(p=0.036). Compared to wt mice, Ph+ ALL mice showed significantly lower miR-142-3p level in all the immunophenotypically identified BM lymphoid subpopulations, including progenitor B (pro-B, B220+CD19+CD43+IgM-,~19.1-fold lower,p<0.0001), precursor B (pre-B, B220+CD19+CD43-IgM-,~9.7-fold lower, p<0.0001), and other immature B (B220lowCD19+CD43-IgM+, ~2.4-fold lower, p<0.001) cells, except for mature B (B220highCD19+CD43-IgM+) cells. Ph+ ALL mice exhibited a miR-142-3p gradient expression pattern following the lymphoid differentiation hierarchy, with the lowest levels found in the pro-B and pre-B populations. These results prompted us to hypothesize that, loss of miR-142 may contribute to primitive B cell expansion possibly due to B cell differentiation blockage in Ph+ ALL mice. To prove this, we crossed miR-142 double knock-out (d-ko)mice with Ph+ ALL mice to generate miR-142(ko)Tg(P190-BCR/ABL) mice. Homozygous miR-142(d-ko)Tg(P190-BCR/ABL) mice were not viable due to an overly aggressive leukemia phenotype. Heterozygous miR-142(wt/ko)Tg(P190-BCR/ABL) mice had evidence of more rapid expansion of pro-B cells in blood(PB; 47.9% vs. 9.8%, p<0.0001), BM (48.2% vs. 13.2%, p<0.01)and spleen(32.3%vs. 4.4%, p<0.01) at 6 weeks old and a significantly reduced survival(median survival 44 vs.80 days, p<0.0001), compared to miR-142(wt/wt)Tg(P190-BCR/ABL) controls. BM cells (CD45.2) from miR-142(wt/ko)Tg(P190-BCR/ABL) mice (n=4) or miR-142(wt/wt)Tg(P190-BCR/ABL) mice (n=5) were then transplanted into congenic CD45.1 recipient mice (n=18 and n=15 respectively).Recipients of BM cells from miR-142(wt/ko)Tg(P190-BCR/ABL) donors showed increased engraftment (94% vs. 77% in PB at 4 weeks, p<0.0001) and significantly reduced survival(median survival 25 vs. 49 days, p<0.0001), as compared with recipients of BM cells from miR-142(wt/wt)P190-BCR-ABL mice. Finally, upon ex vitro exposure to the TKI nilotinib (5uM for 48 hours), miR-142(wt/ko)Tg(P190-BCR/ABL) BM cells showed reduced apoptosis (7.0% vs.37.5% vs p<0.05) and increased cell viability (66% vs.16.2%, p<0.05) compared with miR-142 (wt/wt)Tg(P190-BCR/ABL) BM cells. In vivo treatment studies with TKI treatment are ongoing and data will be presented at the meeting. In conclusion, miR-142 downregulation promotes rapid Ph+ ALL growth likely by contributing to a blockage of B cell differentiation, and may mediate resistance to TKIs.

Disclosures
Stein: Celgene: Speakers Bureau; Amgen Inc.: Speakers Bureau. Jin:The National Natural Science Foundation of China: Research Funding; College of Medicine, Zhejiang University: Employment.