Selinexor enhances the effect of venetoclax on Acute Myeloid Leukemia stem and progenitor cells by disrupting lysosomal function Free

AML is a hematologic malignancy that originates from leukemia stem cells (LSCs) which maintain their self-renewal ability by enhancing autophagic activity, a characteristic that renders them resistant to eradication by conventional chemotherapeutic agents. Although venetoclax-based therapies are effective in AML and can eliminate LSCs, some patients still relapse. Exportin 1 (XPO1) is an essential nucleo-cytoplasmic transport protein responsible for the export of various proteins mainly tumor suppressors. Selinexor(SEL), a XPO1 inhibitor, has strong activity against AML-initiating cells and block autophagic flux. Here, we show that cotreatment with SEL and venetoclax (VEN) results in enhanced cell killing in vitro of primary AML and LSCs.

CCK8 and flowcyte were used to examine the interaction of SEL and VEN on LSC-like cell lines (CD34⁺ KG1α and Kasumi-1 cells) and primary CD34⁺CD38⁻ AML cells. RNA sequencing, protein sequencing, tandem fluorescent protein-tagged LC3, immunoblotting, and Acridine orange (AO) staining were employed to explore the molecule mechanism for the cytotoxicity of selinexor with or without venetoclax against LSC-like cell lines. Kasumi-1-GFP cell derived xenografts mouse model were established to investigate the in vivo efficacy of the combined regimen.

We performed 296 drugs to identify effective inhibition in Kasumi-1 cell, and found 73 drugs could achieved an that inhibitors that target BCL-2 family, HDAC, and XPO1. We evaluated the pairwise combinations of SEL, chidamide (CHI), VEN, and azacitidine (AZA) in MV4-11, MOLM13, KG1α, and Kasumi-1 cells using the ZIP model to assess their synergistic effects. Cotreatment with VEN and SEL exhibited the highest ZIP δ synergy scores and demonstrated significant loss of viability and enhanced apoptosis across multiple cell lines, particularly in LSC-like cells. We collected BMNCs from 5 AML patients and PBMCs from 5 healthy donors during mobilization to evaluate the in vitro effects of the combination therapy. Results demonstrated that SEL significantly sensitized VEN to induce apoptosis in CD34+ AML cells and targeted CD34+CD38− LSCs, while having minimal impact on normal CD34+CD38− hematopoietic stem cells (HSCs) . After RNA-seq in Kasumi-1, GSEA revealed that SEL enriched DEGs in the autophagy pathway. Both RNA-seq and whole-proteome sequencing showed that the combination enriched in the lysosome pathway after GSEA analysis, which is also related to autophagy. We validated the effects of SEL/VEN ± CQ on autophagy using Western blotting. The results indicated that VEN promotes both the formation and degradation of autophagosomes, whereas SEL enhances autophagosome formation but inhibits autophagosome-lysosome degradation. To more intuitively observe the effects of SEL + VEN on autophagic flux, we established stable LSC-like cells expressing RFP-GFP-LC3. The results showed that VEN activates autophagic flux, which could be blocked by the addition of SEL. Literature suggests that autophagy activation promotes cell survival and that autophagy and apoptosis are interconvertible. Thus, we used CQ as a positive control to block autophagy and evaluated its impact on VEN-induced apoptosis. Annexin V/PI staining confirmed that CQ, like selinexor, increased venetoclax-induced apoptosis in Kasumi-1 and KG1α cells. Immunoblot analysis revealed that CQ enhanced venetoclax-induced apoptosis, as evidenced by increased levels of cleaved caspase-3 and cleaved PARP1 in Kasumi-1 and KG1α cells. SEL blocked the activation of autophagic flux caused by VEN, downregulated the expression of LAMP1 (lysosomal associated membrane protein 1) and induced the dysfunction of lysosomes, thereby preventing the degradation of autophagosome, and ultimately promoted cell death. Additionally, in the Kasumi-1 xenograft mouse model, combination therapy demonstrated significantly enhanced antitumor efficacy compared to each monotherapy. Besides, the combination improved mouse survival without lethal toxicity. These data illustrate the application prospects of combined oral administration of VEN and SEL for the treatment of LSC-like cells.

Our studies reveal synergy with selinexor and venetoclax in acute myeloid leukemia and provide a rationale for eliminating leukemia stem cells. A single-arm prospective clinical study evaluating selinexor combined with venetoclax efficiency and safety in elderly, unfit AML patients is undergoing (IIT 2022132).