Inhibition of Lysyl Oxidases Synergizes with 5-Azacytidine to Restore Erythropoiesis in Myeloid Neoplasms

Abstract

Introduction: Limited response rates and frequent relapses during standard of care with hypomethylating agents (HMAs) in myeloid neoplasms (MN) require urgent improvement of this treatment indication. The altered bone marrow (BM) niche in MN is increasingly being recognized as a perspective therapeutic target. Upregulated lysyl oxidase (LOX/LOXL) activity in MN BM contributes to the deposition of aberrant extracellular matrix (ECM). Here, we show in pre-clinical models of MN that the inhibition of niche-associated LOX/LOXL activity in combination with 5-Azacytidine (5-AZA) restores erythropoiesis and reduces disease burden in BM samples of anemic patients with MN. Methods: Primary co-cultures of BM mesenchymal stromal cells (MSCs) and hematopoietic stem and progenitor cells (HSPCs) were established from n=31 anemic MN patients and treated with the pan-LOX/LOXL inhibitor PXS-5505, 5-AZA or PXS-5505 + 5-AZA combination (P+A) for 4 days. The differentiation potential of HSPCs in this co-culture was assessed by colony forming unit (CFU) assays and flow cytometry. In some of the experiments, MSC monolayers were pre-treated with PXS-5505 for 7 days and decellularized to obtain ECM. Subsequently, autologous BM HSPCs were cultured on ECM in the presence of 5-AZA or P+A. Patient-derived xenografts (PDX) were established in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice and treated with 3 cycles of 5-AZA, PXS-5505 (30 mg/kg on alternate days) or the combination of both (P+A). Results: MSCs of MN patients significantly overexpressed LOX/LOXL transcripts as compared to healthy age-matched controls. Remarkably, based on the CFU assay the combination of 5-AZA with the pan-LOX/LOXL inhibitor PXS-5505 improved erythroid differentiation of patient HSPCs that poorly responded to 5-AZA monotherapy. From a total of n=31 anemic patients with advanced-stage myelodysplastic syndromes, myeloproliferative neoplasia and chronic myelomonocytic leukemia, n=20 patients (65%) displayed an erythroid response to P+A combination. Of these, n=11 patients (35%) responded exclusively to P+A, whereas n=9 patients (29%) also concomitantly responded to 5-AZA monotherapy. The comparison of P+A versus 5-AZA monotherapy for improving erythroid response showed a relative risk of 0.45 with 95% confidence interval 0.24-0.80 (p=0.0103). Of note, P+A treatment facilitated differentiation of CD235a+ erythroid progenitors from HSPCs sub-clones with low mutational variant allele frequencies (VAFs). The mechanism of action underlying this drug combination required direct cellular contact of HSPCs with BM stroma components including MSCs and ECM, and is dependent on integrin signaling. Therefore, this novel drug combination represents a genuinely BM microenvironment targeting therapy. We further confirmed these results in vivo using a BM niche-dependent PDX mouse model engrafted with human MN. P+A treatment favored erythroid differentiation of human HSPCs in vivo, showing the directly improved human erythropoiesis in the total BM of PDX mice as well as increased erythroid production after human erythropoietin induction ex vivo. In addition, P+A treatment induced the greatest reduction of disease burden as assessed by decrease of spleen indices, total human engraftment rates, BM fibrosis grades and mutational VAFs. Conclusions: The response to 5-AZA in MN can be potently augmented by concomitant LOX/LOXL inhibition. These results lay out a strong pre-clinical rationale for efficacy of combination treatment of PXS-5505 with 5-AZA especially for anemic and red blood cell transfusion-dependent MN patients, which will be assessed in forthcoming clinical trials.