XPO1 Overexpression Is a Mechanism of Resistance to Eprenetapopt and 5-Azacitidine Therapy That Can be Therapeutically Exploited for the Treatment of TP53 Mutated Myeloid Malignancies

TP53 mutated myeloid neoplasms (TP53-MNs) are among the most aggressive and chemo-refractory adult leukemias. Even after allogeneic stem cell transplant, the median survival for this genetic subset is less than one year. Therefore, an enormous unmet need exists to develop novel therapeutic strategies and understand resistance mechanisms to existing therapies. In a phase 1/2 clinical trial that combined eprenetapopt with 5-azacitidine in TP53 mutated myelodysplastic syndrome, we observed promising activity with a 71% overall response rate, 50% complete response rate, and 47% molecular remissions. However, relapse inevitably occurred with the emergence of the same pretreatment TP53 mutation without secondary mutational events suggesting that resistance is not due to the acquisition or expansion of subclonal mutations. To identify mechanisms responsible for eprenetapopt and 5-azacitidine resistance, we performed a genome-wide CRISPR screen in MOLM13 leukemia cells which were CRISPR-Cas9 edited to harbor an endogenous TP53^R175H mutation and treated with eprenetapopt and 5-azacitidine, 5-azacitidine alone, or vehicle control. After 21 days of treatment, the highest sgRNAs de-enrichment score targeted the nuclear exportin XPO1 suggesting that XPO1 is an essential survival gene after eprenetapopt and 5-azacitidine treatment (Figure 1A, p=0.0007). We confirmed our CRISPR screen results by generating XPO1 deleted single clones in TP53^R175H mutated MOLM13 and K562 leukemia cells and demonstrated that XPO1 KO cells are more sensitive to eprenetapopt and 5-azacitidine treatment. To determine whether XPO1 is a biologically relevant resistance mechanisms in human leukemia, we generated resistant MOLM13 and K562 TP53^R175H cells by daily treatment of stepwise increasing concentrations starting at the IC₅₀ for each compound alone and in combination. The resultant 5-azacitidine and combination resistant cells were observed to upregulate XPO1 protein and mRNA levels. The clinical relevance of this putative resistance mechanism was additionally explored with single cell RNA sequencing of primary bone marrow samples to determine hematopoietic stem and progenitor (HSPC) specific XPO1 expression in relapsing and responding patients (n=3/grp) on therapy. This revealed an increase in XPO1 expression in relapse compared to responding samples across all stem and myeloid HSPCs evaluated.

Because eprenetapopt has been demonstrated to bind and refold p53 and XPO1 is responsible for shuttling wild type p53 to the cytoplasm, we hypothesized that refolded XPO1 dependent p53 cytoplasmic shuttling may be a mechanism of resistance to this combination therapy. To that end, resistant TP53^R175H mutated MOLM13 cells demonstrated increased levels of refolded cytoplasmic p53 protein expression measured by immunofluorescence compared to cells acutely treated with eprenetapopt and 5-azacitidine that was rescued by treatment with the XPO1 inhibitor leptomycin B. Additionally, we evaluated nuclear and cytoplasmic p53 protein levels by immunohistochemistry in serial bone marrow biopsies from the above clinical trial. Using a machine learning algorithm to identify both nuclear and cytoplasmic p53 protein levels we observed that the nuclear to cytoplasmic ratio of p53 was decreased in relapsed but not responding samples (Figure 1B). To determine whether XPO1 can be therapeutically exploited in TP53-MNs, we performed in vitro viability assays with increasing doses of eprenetapopt and the XPO1 inhibitor selinexor and the second generation eltanexor. Eprenetapopt was synergistic with both eltanexor and selinexor in MOLM13 and K562 TP53^R175H leukemia cells and cells resistant to combination therapy. We tested this in vivo utilizing TP53 mutated leukemia cell line xenografts treated with either vehicle alone, eprenetapopt, eltanexor or the combination of eprenetapopt and eltanexor. Combination treatment xenografts had decreased tumor volume compared to vehicle or each single agent alone. Our data collectively suggest that XPO1 is overexpressed after eprenetapopt and 5-azacitidine treatment resulting in re-folded TP53 being shuttled to the cytoplasm leading to therapeutic resistance. Patient derived xenograft assays testing this novel combination therapy in vivo with TP53-MN primary samples are underway and will be presented during the ASH meeting.

Komrokji:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia, Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Taiho Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; Acceleron Pharma: Consultancy; AbbVie: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; CTI BioPharma, Innovent: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Honoraria, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Taylor:Karyopharm, Inc: Honoraria. Sallman:Magenta: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Lixte: Patents & Royalties: LB-100; Incyte: Speakers Bureau; Syntrix Pharmaceuticals: Research Funding; Intellia: Membership on an entity's Board of Directors or advisory committees; Nemucore: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees. Padron:Kura: Research Funding; Incyte: Research Funding; Stemline: Honoraria; Taiho: Honoraria; BMS: Research Funding; Syntrix Pharmaceuticals: Research Funding; Blueprint: Honoraria.