Differential impacts of Lama4 deficient microenvironment on fitness of BCR::ABL1-expressing cells during chronic myeloid leukemia initiation and progression Free

Introduction: Chronic myeloid leukemia (CML) is a clonal stem cell disease driven by BCR::ABL1 fusion gene. It has been proposed that the BCR::ABL1 clone fitness and evolution determine CML initiation, progression and therapy responses even though not much is known about how the clone fitness is regulated, limiting opportunities to identify prognostic markers and new effective targeted therapy for CML. Evidence suggests microenvironmental impacts on leukemia clone fitness during the disease development. However, the molecular mechanisms remain poorly defined. We have found that the laminin alpha 4 chain (LAMA4), a functional chain of several laminin isoforms, is exclusively expressed by bone marrow (BM) stromal cells including mesenchymal stem cells (MSC) and its loss in host niche promotes acute myeloid leukemia progression and therapy resistance in mice (Cai, et al., Blood, 2022). Additionally, LAMA4 is reduced in the BM of chronic phase CML MSCs (Dolinska, et al., Blood, 2023). Nevertheless, the impact of LAMA4 expression on CML progression and clone fitness remains unexplored.

Methods: We have determined the impact of LAMA4 on CML clone fitness during the disease initiation and therapy responses using the inducible CML mouse model (SCL×tTA×TRE-BCR::ABL1) and BM aspirates from newly diagnosed CML patients (N=5). The BCR::ABL1 clone size and CML onset in the SCL×tTA×TRE-BCR::ABL1 mice crossed with Lama4^-/- and Lama4^+/+ mice were monitored by droplet digital PCR (ddPCR) at 2-6 weeks after CML induction by tetracycline withdrawal. To decipher the effect of Lama4^-/- niche on CML clone fitness (growth/survival), we evaluated cell cycle status of hematopoietic stem and progenitor cells (HSPC, LIN^-SCA1⁺KIT⁺) in the mice by simultaneous staining of KI67 and DNA at 2 weeks post-tetracycline withdrawal. The therapeutic effect of LAMA4 proteins on human CML stem cells was tested by treating patient BM CD34⁺CD38^- cells with recombinant LAMA4 peptides in cocultures using primary CML patient-derived BM MSCs, followed by cobblestone area forming cells (CAFC) and long-term culture-initiating cell (LTC-IC) assays and ddPCR for residual BCR::ABL1 clones.

Results: Firstly, we observed a delayed CML onset and prolonged survival of Lama4^-/-×BCR::ABL1 mice, compared to Lama4^+/+×BCR::ABL1 mice, suggesting the suppressive effects of Lama4 deficient niche on CML initiation and progression. The effects were supported by reduced BCR::ABL1 clone size in Lama4^-/- mice, detected by ddPCR of the relative fractions of BCR::ABL1-expressing mononuclear cells in blood (p=0.025) and myeloid progenitors including megakaryocyte erythroid progenitors (p=0.01) and common myeloid progenitors (p=0.057) in Lama4^-/-×BCR::ABL1 mouse spleen compared to that in Lama4^+/+×BCR::ABL1 mice at 2 weeks post tetracycline withdrawal. The slower initiation of CML in the Lama4^-/-×BCR::ABL1 mice was accompanied with decreased frequencies of LSC (LSKCD150⁺) in the BM (p=0.029) as well as in the spleen (p=0.014) at week 2 post CML induction. Consistent with this, cell cycle analysis revealed an increased quiescent fraction of LSK cells in Lama4^-/-×BCR::ABL1 mice, reflected in thehigher proportion of the cells in G0/G1 status, indicating that Lama4 deficient environment suppresses CML HSPC proliferation. Surprisingly, treatment with recombinant LAMA4 peptides inhibited CML patient BM CD34⁺CD38^- cell growth in CAFC assay (p=0.001, N=4), and even more strikingly (about 80% inhibition) in LTC-IC stem cell assay while imatinib did not show any clear inhibition. No clear synergy was observed between LAMA4 and imatinib. The residual CML clones (BCR::ABL1⁺) were comparable in all treatment groups while being lower than that in the untreated mononuclear cells, suggesting LAMA4 peptides inhibited CML stem cells by reducing BCR::ABL1 clone fitness. Notably, the inhibition was selective on CML stem cells, since no effect was observed on healthy donor BM CD34⁺CD38^- cells (N=3).

Conclusions: Our data suggested differential impacts of Lama4 deficiency on the fitness of BCR::ABL1-expressing cells during CML initiation and therapy response. We observed a suppressive effect on CML clone growth of Lama4 deficient niche during CML initiation, while better fitness of the CML stem cells in in vitro environment with reduced LAMA4 levels. Together, our work suggested opposing roles of Lama4 deficient niche for CML cell fitness during CML initiation and progression.