BMP Pathway Alterations in Chronic Myelogenous Leukemia Lead to the Amplification of Primitive Progenitors Output in Chronic Phase
Authors: Nicolini Franck E. , Laperoussaz Bastien , Jeanpierre Sandrine , Sagorny Karen , Kaniewski Bastien , Morisset Stephane , Salesse Stéphanie , Voeltzel Thibault , maguer-Satta Véronique 
Published: October 2019

Abstract
Abstract 1663
The presence of inherently resistant Ph+ leukemic stem cells (LSC) to tyrosine kinase inhibitors (TKI) in chronic phase chronic myelogenous leukemia (CP CML) might be responsible for the long-term disease persistence, requiring the indefinite prolongation of TKI, and represent a reservoir for the initiation of an overt resistance. Despite quiescence, these Ph+ LSC maintain high level interactions with their microenvironment and might display some alterations to different pathways to survive, responsible for disease persistence. In this perspective, we studied some actors involved in the maintenance of the stem cell niche and normal hematopoiesis (Jeanpierre S. et al. Blood 2008), the bone morphogenetic proteins (BMP-2 and BMP-4) in a series of 45 CP CML patients harvested at diagnosis and compared the results to that of 15 normal bone marrow (BM) counterparts from healthy donors. BMPs are members of the TGF-b family, highly secreted by the normal BM stem cell niche, that regulate numerous cellular events such as proliferation, differentiation, migration, adhesion and apoptosis in various cellular systems including hematopiesis. The deregulation of these events is the hallmark of CP CML and they are all involved in the CML disease phenotype in CP. All CML and BM samples were CD34+ purified. RQ-PCRs on total cells, CD34− and CD34+ progenitors for the different members of the BMP pathway show specific alterations in CML: in the CD34+ primitive compartment, BMP-4 itself is downregulated as well as Bambi, Smad1, Smad5, and Runx2 while Smad6 is specifically upregulated. Interestingly, while analyzing the CD34+ and CD34− compartments from individual patients, we observed a differential expression of the signaling pathway inhibitors Smad6 and Follistatin (low levels in leukemic CD34−, high levels in leukemic CD34+ cells when compared to their normal purified counterparts), and BMP-2, BMP-4, and three of the BMP specific receptors (BMP-RIa, BMP-RIb and BMP-RII) are significantly and selectively downregulated in CD34+ CML progenitors. We further characterized the impact of 7-days in vitro exposure to BMP-2 and BMP-4 in 4GF (IL-6, SCF, IL-11, IL-3) serum-free conditions of CD34+ CML and normal BM progenitors using immunophenotypic, CFC and LTC-IC assays. The functional assays results suggest in responding patients, that BMP-4 is able to selectively amplify the genotyped Ph+ LTC-IC compartment (while BMP-2 partly maintains it, whereas TPO amplifies normal Ph− LTC-IC as well as Ph+ LTC-IC) as mentioned in figure 1.
Figure 1: Total LTC-IC-derived week 5 colonies per 1000 seeded cells within the responding CML samples. Black bars are CML samples and white bars are normal BM counterparts, (*: p<0.05). Figure 1:. Total LTC-IC-derived week 5 colonies per 1000 seeded cells within the responding CML samples. Black bars are CML samples and white bars are normal BM counterparts, (*: p<0.05).
The CFC compartment was highly amplified when exposed to either BMP-2 or BMP-4 (but not TPO) when compared to normal BM progenitors as controls. In addition, within responding patient samples, only BMP-4 significantly amplified the number of immature cells as compared to TPO or BMP-2 treated cells. Single cell experiments in serum-free conditions with sorted CD34+38− chronic phase CML cells from diagnosis, in parallel with normal BM cells, in the presence or not of BMP-2 & −4 have been performed. A high cloning efficiency was observed with CML cells (∼17%), compared to that with normal BM counterparts (3%) as usual, and in addition, BMP-2 seemed to promote erythroid commitment of primitive CML cells, while BMP-4 increased CFU-GM and CFU-GEMM output.
In conclusion, these data provide some evidences that the BMP pathway is selectively altered in primitive CP CML progenitors at diagnosis, and its molecular and functional alterations might be involved in the disease phenotype. Whether this is a cause or a consequence of the disease and its involvement in stem cell persistence on TKI is currently under examination.

Disclosures:
Nicolini: Novartis: Research Funding. Sagorny:BMS: Research Funding. maguer-Satta:Novartis: Research Funding.