The selective PI3Kδ inhibitor roginolisib synergizes with ruxolitinib against progenitor cells from naïve and JAK inhibitor-refractory/resistant patients with myelofibrosis

Abstract BACKGROUND. Following demonstration of abnormally activated JAK2/STAT signalling caused by driver mutations in JAK2, CALR, MPL, JAK inhibitors (JAKi) were developed and approved for patients (pts) with myelofibrosis (MF). However, other intracellular pathways, such as the PI3K/AKT/mTOR, are implicated in abnormal cell proliferation and the inflammatory milieu of MF pts, and may contribute to mechanisms of resistance/refractoriness (R/R) to JAKi. PI3K inhibitors were used in clinical trials in MF with evidence of activity but also showed significant toxicities that prevented further development. Compared to those pan PI3K inhibitors, isoform-selective PI3K inhibitors are now being developed that may have less toxicity. Roginolisib (IOA-244) is a first-in-class, non-ATP-competitive, PI3Kδ inhibitor currently undergoing clinical trials in solid and hematologic malignancies. AIM. We investigated the activity of IOA-244, either alone and in combination with JAKi, in cellular models and primary hematopoietic cell progenitors from pts with MF. RESULTS. We first evaluated the anti-proliferative activity of IOA-244 (0-10 µM) on JAK2V617F-mutated UKE-1 cell line over 48hr, both alone and in combination with Ruxolitinib (Ruxo, 0.05-0.1 µM). IOA-244 alone failed to inhibit cell growth, even at the highest dose, compared to IC50 of Ruxo being 0.45 ± 0.02 µM. Combining IOA-244 (0.5-3 µM) with suboptimal Ruxo doses significantly inhibited cell growth compared to untreated cells or either drug alone. The best inhibition value ranged from 43.9% ± 4.2% (at 1µM IOA-244 + 50nM Ruxo) to 50% ± 1.3% (at 1µM IOA-244+100nM Ruxo). All combinations showed statistical significance (p<0.01 vs. controls; p<0.05 vs. IOA-244 and Ruxo alone). Chou-Talalay analysis confirmed synergy (CI<1) of the 2 drugs across all dose combinations, ranging from CI 0.23 to 0.53. Overexpression of PI3Kδ by Western blot was demonstrated in CD34+ cells from MF pts (naïve and Ruxo-treated), unlike in cord blood CD34+. We then performed clonogenic assays for granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells on CD34+ cells from 9 MF pts (5 treatment-naïve, 4 Ruxo R/R). IOA-244 significantly inhibited colony formation, yielding IC50 values of 4.7 ± 1.4 µM for CFU-GM and 5.9 ± 1.3 µM for BFU-E. We observed a significantly higher responsiveness in treatment-naïve patients; corresponding IC50 values were considerably lower for both CFU-GM (1.4 ± 0.6 µM) and BFU-E (3 ± 0.9 µM) when compared to R/R patients, who showed IC50s of 9.4 ± 0.5 µM and 8.7 ± 1.1 µM, respectively (p<0.001 for both). Combining suboptimal doses of IOA-244 (0.5-1 µM) and Ruxo (50-100 nM) synergistically inhibited both BFU-E and CFU-GM. Specifically, for CFU-GM, there was a significant reduction, ranging from 69.2% ± 4.7% (with 0.5 µM IOA-244 + 50 nM Ruxo) to 81.1% ± 4.7% (with 1µM IOA-244 + 100nM Ruxo). Similarly, BFU-E colony formation was also notably reduced, with inhibition ranging from 67.2% ± 3.9% to 81.5% ± 3.1% using these same combinations. All combinations exhibited statistically significant synergy (p<0.05), with CI values ranging from 0.25-0.5 for CFU-GM and 0.36-0.5 for BFU-E. We also evaluated combinations of suboptimal IOA-244 (0.5-1 µM) and Momelotinib (Mome, 5-50 nM) doses. All tested combinations significantly reduced colony formation compared to controls and single agents. For CFU-GM, reductions ranged from 72.6% ± 1.5% (with 0.5 µM IOA-244+5 nM Mome) to 78% ± 1.2% (with1 µM IOA-244+50 nM Mome). Similarly, BFU-E reductions varied from 64.2% ± 0.9% to 74.6% ± 1.3% across those combinations. All combinations exhibited statistically significant synergy (p<0.05), with CI values ranging from 0.07 to 0.3 for CFU-GM and 0.08 to 0.26 for BFU-E. Finally, to ascertain the known IOA-244's anti-inflammatory effects, we treated MF mononuclear cells with increasing doses (0, 0.5 and 1 µM) for 3 hours. Real-time PCR analysis showed a significant decrease in the mRNA of 10 out of 30 cytokines. At the maximal dose, reductions were statistically significant (p<0.05) for IL-10 (-53%), IL-18 (-41.3%), IL-1β (-52.5%), IL-2 (-49.2%), IL-6 (-76.1%), IL-8 (-58.8%), TNF (-44.3%), CCL1 (-67.9%), CCL4 (-39.8%), and CXCL10 (-84.2%). CONCLUSIONS. These findings support efficacy of IOA-244, a selective PI3Kδ inhibitor, in primary MF cells, especially when combined with JAK2 inhibitors, providing a rationale for the ongoing phase 2 HEMA-MED clinical trial (NCT06887803).