Selinexor Depletes Ruxolitinib Refractory Myelofibrosis Hematopoietic Stem Cells By Inducing Apoptosis and Blunting the Pro-Inflammatory Milieu
Authors: Lu Min , Mia MD Babu , Xia Lijuan , Ellero Andrea , Walker Christopher J. , Hoffman Ronald 
Published: December 2024

Introduction

Myelofibrosis (MF) originates at the level of the hematopoietic stem cell (HSC). JAK2 inhibitor (JAK2i) therapy represents the standard of care for MF patients, but only modestly prolongs overall survival due to its inability to deplete MF HSCs. Furthermore over 70% of MF patients become intolerant or refractory to ruxolitinib (RUX) therapy after 3 years of treatment. Selinexor (SEL), a first-in-class oral selective XPO1 inhibitor, in combination with RUX has resulted in rapid, deep, and sustained spleen and symptom responses in JAKi-naïve MF patients (Tantravahi et al, ASH 2023) which prompted us to examine the effects of SEL on MF HSCs. Selinexor has anti-cancer activity by sequestering tumor suppressor proteins in the nucleus including p53 and others. Previously, we reported that SEL induces apoptosis of both TP53 wild-type (WT) (UKE-1) and TP53 mutant (mut) (SET-2) MPN cell lines and selectively reduced the absolute number of JAK2V167F+ colonies assayed from MF patient CD34+ cells irrespective of their TP53 status (Lu et al, ASH 2023). To further investigate the therapeutic potential of SEL to deplete RUX refractory (RR) MF HSCs and its mechanism of action (MOA), we used a humanized xenograft mouse assay system and performed bulk RNA sequencing (RNAseq).

Methods

In vivo stem-cell targeting study: RR MF CD34+ cells were treated with SEL at 200 nM in SFEM medium with SCF, TPO and FLT-3L for 3 days. Cells were collected and transplanted into sub-lethally irradiated NSG-SGM3 mice. After 12 weeks, the mice were sacrificed, and the degree of human cell chimerism in the bone marrows (BM) and spleens was assessed by flow cytometry.

Bulk RNAseq analysis: SET-2 cells, UKE-1 cells, primary RR MF CD34+ cells (n=3, TP53 WT patients, 2 patients JAK2V617F, 1 patients triple-negative), and healthy donor (HD) CD34+ cells (n=3) were treated with SEL at 200nM and 500 nM for 4 hours then RNAseq was performed.

Results

Pre-treatment with SEL for 3-days decreased total RR MF CD34+ cell numbers by 70%; FACS analysis showed a 20% increase in Annexin V+/CD34+ cells, indicating a SEL-induced apoptosis, the results consistent with our previous observations. Mice transplanted with mock-treated MF CD34+ cells experienced a 42% increasing in spleen weight as compared with normal non-transplanted mice (p=0.003, n=5), while, mice transplanted with SEL-treated compared with mock-treated MF CD34+ cells experienced a 31% reduction in spleen weight (p=0.014, n=5). The degree of human cell chimerism (hCD45+ cells) was significantly reduced in both the BMs and spleens of mice receiving SEL-pretreated RR MF CD34+ cells (p=0.024 and p=0.004, respectively, n=5 in each group). Furthermore, human CD34+ cell numbers were significantly reduced in the spleens of mice receiving SEL-pretreated CD34+ cells (p=0.008). The degree of MF CD41+ cell chimerism was also significantly decreased by &gt;90% in spleens (p=0.003) and by 30% in the BM of SEL-pretreated recipient mice. These findings indicate that SEL depletes MF HSCs from RR MF patients.

SEL modulated key MF-pathways in a dose dependent manner in both TP53WT and TP53mut cell lines, including upregulation of TP53, TP63, and TP73 transcripts in UKE-1 and the upregulation of p63 and p73 in SET-2 cells as shown by qPCR.

RNAseq was used to examine the MOA of SEL on RR MF CD34+ cells. Compared to HD CD34+ cells, RR MF CD34+ had a significant upregulation of inflammatory and proliferative pathways (NFkB, TGFβ and inflammatory response). SEL treatment of MF CD34+ cells significantly downregulated genes involved in inflammation and proliferation, including the TNFα-NFκB (q = 5.44 x 10-13), TGFβ (q = 6.86 x 10-05), IFN-γ (q = 2.13 x 10-02), MYC (q = 5.22 x 10-20), G2M (q = 1.10 x 10-28) and IL-6-JAK-STAT3 signaling pathways (q = 1.63 x 10-04). Notably, even at the lowest tested dose (200 nM), SEL selectively upregulated TP53 signaling (q = 9.03 x 10-5) and apoptotic pathways (q = 0.008) in RR MF samples as compared to similarly treated HD cells.

Conclusions

The humanized mouse model studies demonstrated that SEL treatment effectively targets not only MF progenitor cells but also stem cells from RR patients. SEL selectively induced apoptosis and suppressed multiple proliferative and oncogenic pathways in RR MF CD34+ cells which have been implicated in MF stem cell fitness. These findings suggest that SEL is a potential therapeutic agent capable of depleting RR MF HSCs by affecting multiple intracellular pathways.