BCR-ABL1 Kinase Activity but Not Its Expression Is Dispensable for Ph+ Quiescent Stem Cell Survival Which Depends on the PP2A-Controlled Jak2 Activation and Is Sensitive to FTY720 Treatment
Authors: Neviani Paolo , Harb Jason G , Oaks Joshua J , Walker Christopher , Santhanam Ramasamy , Paisie Carolyn , Eiring Anna , Zhang Bin , Perazzona Bastianella , Ma Y. , Mao Charlene , Marcucci Guido , Holyoake Tessa L. , Volinia Stefano , Cortes Jorge E. , Caligiuri Michael A. , Huettner Claudia , Bittman Robert , Chen Ching-Shih , Arlinghaus Ralph B. , Hokland Peter , Roy Denis-Claude , Bhatia Ravi , Perrotti Danilo 
Published: October 2019

Abstract
Abstract 515
The success of tyrosine kinase inhibitors as first line therapy for t(9;22) Chronic Myelogenous Leukemia (CML) depends on the addiction that Philadelphia-positive (Ph+) hematopoietic progenitors, but not quiescent Ph+ stem cells, have for BCR-ABL1 tyrosine kinase activity. We reported that the activity of the tumor suppressor PP2A is inhibited in a SET-dependent manner in CML progenitors and CD34+/CD38- stem cells from chronic phase (CP) and, to a greater extent, blast crisis (BC) CML patients (Neviani P. et al.: Cancer Cell 2005, J. Clin. Invest 2007, and ASH 2008). Restoration of PP2A activity by the immunosuppressive sphingosine analogue FTY720 markedly decreases the number of Ph+ but not normal long-term culture-initiating cells (LTC-IC) and quiescent stem cells (CFSEMAX/CD34+) by suppressing the BCR/ABL kinase-independent enhancement of b-catenin expression/transcriptional activity (Oaks JJ., et al., ASH 2009).
Here we report that FTY720 induces apoptosis of Ph+ CD34+/CD38- cells independent from its phosphorylation as treatment with a phosphorylated FTY720 did not alter the number of Ph+ CFSEMAX/CD34+ cells. By contrast, two non phosphorylatable and non immunosuppressive FTY720 derivatives did significantly affect survival of Ph+ stem/progenitor cells.
Interestingly, we also noted that the activity but not expression of BCR-ABL1 is considerably lower in quiescent CFSEMAX/CD34+ than CFSE+/CD34+ cells that underwent at least one division (∼80% decrease; n=3). Conversely, BCR-ABL1 expression is significantly higher in quiescent than proliferating CFSE+/CD34+ cells, suggesting that BCR-ABL1 might serve as a scaffold for other kinase(s) able to sustain survival and quiescence of Ph+ stem cells. Indeed, we found that expression of the K1172R kinase-deficient BCR-ABL1 mutant enhances expression and activity of Jak2, a kinase that is not only associated with BCR-ABL1 but is also capable of inactivating and being inactivated by PP2A. Accordingly, lentiviral shRNA-mediated BCR-ABL1 downregulation in Ph+ CD34+/CD38- stem cells resulted in marked (≥70% inhibition, P<0.05) reduction of Jak2 activity (measured by Jak2pY1007/8 intracellular flow cytometry) and of the PP2A inhibitor SET (≥20% inhibition, P<0.05). Consistent with a role of Jak2 as regulator of Ph+ stem cell survival, pharmacologic Jak2 inhibition (Jak2 inhibitor 1, 1uM; TG101209, 2.5uM; or TG101348, 1uM and 0.5uM) significantly reduces the number of Ph+ CFSEMAX/CD34+ (47% reduction, n=4), impairs LTC-IC Ph+ colony output (60% reduction, n=4), and induces apoptosis of Ph+ CD34+/CD38- cells. Inhibition of Jak2 also resulted in impaired b-catenin dependent transcriptional activity (61% reduction by LET-TCF luciferase assay, n=2), suggesting the existence of an active Jak2-PP2A interplay likely controlling survival and self-renewal of Ph+ stem cells through the Wnt pathway. In this regard, in vivo administration of FTY720 (4 weeks; 10mg/Kg/day) to FVB/N recipient mice transplanted with either GFP-sorted 2×106 whole bone marrow or 3×103 Lin−/Sca-1+/c-Kit+ (LSK) stem cells from leukemic scl-tTA/BCR-ABL/GFP mice not only significantly decreased the number of leukemic common and granulocyte-macrophage (CMP and GMP) progenitors but, more importantly, it resulted in 70–80% reduction in the number of the long-term hematopoietic stem cells (Lin−/Sca-1+/c-Kit+/CD48+/CD150−/Flt3−) compared to untreated leukemic mice. Furthermore, severely impaired engraftment (∼90% untreated vs. ∼37% FTY720-treated) was observed in secondary recipients transplanted with 2×106 bone marrow of FTY720-treated leukemic mice, suggesting that FTY720-induced PP2A activation inhibits the ability of BCR-ABL1-expressing LSK cells to undergo extensive self renewal in primary recipients.
Thus, expression but not activity of the oncogenic product of the t(9;22) translocation is important for recruiting and allowing SET-mediated inhibition of PP2A and activation of Jak2; two events important for Ph+ stem cell survival and self renewal. Moreover, the ability of FTY720 and of its non-immunosuppressive derivatives to induce apoptosis of Ph+ progenitors and Ph+ but not normal quiescent stem cells emphasizes the notion that FTY720 and its derivatives represent strong and non-toxic anti-leukemic agents potentially useful not only for the treatment but, perhaps, for eradicating Ph+ leukemias.

Disclosures:
Holyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.