Combining CDK4/6 inhibitor with TMZ and radiation alters cell states for synergistic responses in orthotopic DIPG models

Background: To develop effective therapies for DIPG at different clinical stages, we examined efficacy and mechanisms of action of a combination therapy-abemaciclib (a CDK4/6 inhibitor), temozolomide (TMZ), and radiation (XRT)-in two patient-derived orthotopic xenograft (PDOX) models derived from treatment-naïve (IBs-9119DIPG) and autopsied (IBs-A0317DIPG) tumors. Methods: In vitro synergistic anti-tumor activities were examined in tumor organoids, and in vivo efficacy in the PDOX models treated with abemaciclib (75 mg/Kg × 14 days), TMZ (50 mg/Kg × 5 days) and XRT (2 Gy/day × 5 days) alone and in combination (n=10/group, 60 mice/model). Changes of animal survival times were analyzed with log-rank analysis. Mechanisms of treatment response and resistance were elucidated by immunohistochemistry and scRNA-seq analysis. Results: The triple therapy generated synergistic anti-tumor effects in organoids and significantly extended survival times in both PDOX models ( P <0.05) despite their strong cellular state differences. scRNAseq identified reduction of oligodendrocyte-progenitor-like (OPC-like) cells in both models and astrocyte-like (AC-like) cells in IBs-A0317DIPG as response mediators; and revealed expansion of neural progenitor-like (NPC-like) cells in IBs-A0317DIPG and of mesenchymal-like (MES-like) and mitotic-like cells in IBs-9119DIPG as resistance contributors. Pseudotime trajectory analysis uncovered the exit of stemness into differentiation in oligodendrocyte-progenitor-like (OPC-like) cells as a novel mechanism of resistance in the treatment-naïve IBs-9119DIPG, in contrast to the enrichment of stem-like cells in the recurrent model IBs-A0317DIPG. A radiation-resistant subpopulation with novel candidate targets ( NPAS3, TBC1D5, INPP4B ) was also discovered. Conclusions: This study demonstrated strong anti-DIPG capacities of the triple therapy in both untreated and recurrent DIPG tumors by acting on distinct cellular and molecular targets, and identifies previously unrecognized mechanisms underlying DIPG therapy response and resistance.