Specific and Targetable Interactions with the Bone Marrow Microenvironment Govern Outcome in Imatinib-Resistant Chronic Myeloid Leukemia
Authors: Kumar Rahul , Marten Maximilian , Minciacchi Valentina , Pereira Raquel , Zanetti Costanza , Hyduk Nina , Oellerich Thomas , Kvasnicka Hans Michael , Spencer Joel , Schnuetgen Frank , Lin Charles , Lo Celso Cristina , Meister Melanie , Nicolini Franck Emmanuel , Krause Daniela S. 
Published: October 2019

Abstract
Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukaemia (CML), can lead to resistance to tyrosine kinase inhibitors and some are associated with clinically more aggressive disease and worse outcome (Branford et al., 2003).
This phenomenon was faithfully recapitulated in our murine transduction/transplantation model of BCR-ABL1+ CML, in which recipients of bone marrow transduced with the imatinib-resistant BCR-ABL1 point mutant T315I (or Y253F) had shortened survival, compared to wild-type BCR-ABL1. Consistent with the situation in humans we found an increase in the number of blast-like cells and alterations of the immunophenotypic composition of cells in the bone marrow of murine recipients of BCR-ABL1T315I+ leukaemia-initiating cells (LIC).
To understand the interaction between BCR-ABL1+ LIC and the bone marrow microenvironment (BMM), a complex entity of cellular and acellular constituents, which may influence the course of the disease, we used a combination of confocal and 2-photon intravital microscopy of the murine calvarium. BCR-ABL1T315I + Lin- c-Kit+ Sca-1+ (LKS) cells, which contain the leukaemic stem cell (LSC) fraction in this model, homed closer to osteoblastic cells thanLKS cells expressing native BCR-ABL1 which suggests a differential homing location of BCR-ABL1T315I+ LSC. Indeed, in a transwell migration assay we demonstrated faster migration of BCR-ABL1T315I+ compared toBCR-ABL1+ cells. Other experiments revealed an altered actin cytoskeleton and striking differences in focal adhesion kinase (FAK), a component of integrin mediated focal adhesion sites, in BCR-ABL1T315I+ compared to wildtype BCR-ABL1+ cells. Additionally, in in vivo and ex vivo studies we showed that integrin β3 was overexpressed in BCR-ABL1T315I+ compared to wildtype BCR-ABL1+ cells, and that further overexpression of integrin β3 significantly reduced the tumor load and prolonged the survival of mice with BCR-ABL1+, but especially BCR-ABL1T315I+ CML.
Further, we found another component of the focal adhesion complex, namely integrin linked kinase (ILK) and its phosphorylated form (pS243) to be more highly expressed in BCR-ABL1T315I+ compared to BCR-ABL1+ wildtype cells. ShRNA-mediated knockdown of ILK in BCR-ABL1T315I+ LIC significantly reduced the tumor load and prolonged survival in recipient mice. Inhibition of ILK by the use of a commercially available (non clinical grade) inhibitor (Cpd22) in combination with the tyrosine kinase inhibitor ponatinib prolonged the survival of mice compared to treatment with ponatinib alone, further confirming an important role of ILK in BCR-ABL1T315I+ disease.
Fibronectin is a known interaction partner of integrins in the extracellular matrix of the BMM and, as previously reported, its deposition in the extracellular matrix can be regulated by ILK. We found that the deposition of fibronectin in BCR-ABL1T315I + disease was decreased compared to BCR-ABL1+ wildtypecells. Intrafemoral or intravenous administration of fibronectin, however, led to a significant prolongation of survival in 70% of mice with BCR-ABL1T315I + CML, and knockdown of ILK in BCR-ABL1T315I + LIC led to a replenishment of fibronectin levels in the BMM.
In confirmation of these murine data, bone sections of patients with BCR-ABL1T315I+ CML revealed decreased levels of fibronectin,and Western blot analysis of human BCR-ABL1T315I+ CML cellsshowed increased levels of ILK and pS243ILK compared to the BCR-ABL1+ controls.
In summary, these data suggest that interactions with the BMM via the integrin β3/ILK-mediated signaling pathway may influence leukaemic progression and clinical outcome in BCR-ABL1T315I+ imatinib-resistant CML. Targeting the interaction of BCR-ABL1T315I+ leukemia cells with the BMM in the form of ILK inhibition or administration of fibronectin may offer a beneficial, innovative, adjunct treatment strategy for patients with BCR-ABL1T315I+ CML and, as to be tested, possibly also other leukaemias.

Disclosures
Kumar: European Patent No. 16187926.7: Patents & Royalties: European Patent No. 16187926.7-1401 "FIBRONECTIN FOR USE IN THE TREATMENT OF LEUKEMIA" . Nicolini:Sun Pharma Ltd: Consultancy; Incyte Biosciences: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Krause:Glycomimetics Inc. until 06/2016: Consultancy, Research Funding; Merck KgGA in future: Research Funding; Glycotope: Consultancy; European Patent: Patents & Royalties: European Patent No. 16187926.7-1401 "FIBRONECTIN FOR USE IN THE TREATMENT OF LEUKEMIA" .