Janus Kinase Inhibition By Ruxolitinib Combined with Nilotinib Has Superior Anti-Leukemia Propagating Cells Effect in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia
Authors: Kong Yuan , Wu Yi-Lin , Song Yang , Shi Min-Min , Qin Ya-Zhen , Lai Yue-Yun , Jiang Hao , Jiang Qian , Huang Xiao-Jun 
Published: October 2019

Abstract
Background: Relapse, the major cause of treatment failure in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL), seriously affects the long-term survival of Ph+ALL patients even in the modern era of abelson tyrosine kinase inhibitors (TKIs). Leukemia propagating cells (LPCs), defined by their ability to initiate human leukemia and self-renew in immunocompromised mice, almost equal to leukemia stem cells. Relapse of Ph+ALL may result from the persistence of LPCs. Using an anti-CD122-conditioned NOD/SCID xenograft mouse assay, we recently reported that LPCs are enriched in the CD34+CD38-CD58- fraction in human Ph+ALL(Y Kong… XJ Huang*, et al. Leukemia 2014). Further cohort study demonstrated that Ph+ALL patients with LPCs phenotype at diagnosis exhibited significantly higher cumulative incidence of relapse than the other phenotype group even receiving uniform first-line imatinib-based therapy pre- and post-allotransplant (Y Kong, …XJ Huang*, et al. Bone marrow transplantation 2015). Therefore, it is imperative to identify novel therapeutic strategies based on LPCs to improve the prognosis of Ph+ALL patients.
Aims: To compare the gene expression profiles between the sorted LPCs and other phenotype cells from patients with de nove Ph+ALL. Moreover, to investigate whether selective ABL/JAK2 dual inhibition therapy by nilotinib with ruxolitinib could more effectively eliminate imatinib-insensitive LPCs in vitro as well as in humanized Ph+ALL mice.
Methods: To identify the potential molecular basis involved in LPCs-mediated relapse, RNA-sequence and real-time reverse transcription-PCR (qRT-PCR) were performed to analyze the gene expression profiles between the sorted LPCs (CD34+CD38-CD58-) and other phenotype cells (CD34+CD38-CD58+,CD34+CD38+CD58-, and CD34+CD38+CD58+) from patients with de nove Ph+ALL. In order to assess the effects of the selective ABL and/or JAK2 inhibition therapy by the single or different combinations of imatinib/nilotinib, and/or ruxolitinib on Ph+ALL LPCs, drug-induced apoptosis of LPCs was further investigated in vitro, as well as in vivo using sublethally irradiated and anti-mouse CD122 monoclonal antibody conditioned humanized NOD/SCID mice by intra-bone marrow injection.
Results: RNA-sequence demonstrated that JAK2 was highly expressed in the sorted LPCs compared to the other phenotype cells in patients with de nove Ph+ALL, which was further validated by qRT-PCR. Therefore, we investigated the anti-LPCs effects in the following treatment groups including imatinb, nilotinb, ruxolitinib, imatinb combined with ruxolitinib, nilotinb combined with ruxolitinib, and no treatment control groups. In vitro study, nilotinb combined with ruxolitinib induced significantly higher levels of apoptosis in LPCs than the other treatment groups. In Ph+ALL humanized mouse model, bone marrow and spleen of the recipients were efficiently engrafted with human Ph+ALL cells with an aberrant phenotype similar to that in the donor Ph+ALL patients. The engrafted human cells were derived from the Ph+ALL clone indicated by the presence of BCR/ABL transcripts. At 2 months post drugs intervention, similar high levels of human Ph+ALL engraftment were demonstrated in imatinib-treated mice and no-treatment control mice. As predicted, ruxolitinib alone had no significant anti-LPCs effect in this model, but it prevented Ph+ALL engraftment more significantly when administered with nilotinib. Consistent with our flow cytometry data, hematoxylin-eosin staining and immune histochemistry with rabbit anti-hCD34 and CD19 demonstrated that the infiltrating levels of the transplanted LPCs cells were significantly lower in brain, liver, kidney and spleen tissues of nilotinb combined with ruxolitinib-treated mice than the other groups' mice.
Summary/Conclusion: JAK2 was more highly expressed in the sorted LPCs than the other phenotype cells in patients with de novo Ph+ALL. Selective ABL/JAK2 dual inhibition therapy by nilotinib with ruxolitinib could more effectively eliminate imatinib-insensitive LPCs both in vitro and in humanized Ph+ALL mice. These data indicate that selective ABL/JAK2 dual inhibition therapy by nilotinib with ruxolitinib represents a promising anti-LPCs therapeutic approach for patients with Ph+ALL.

Disclosures
No relevant conflicts of interest to declare.