Combining abemaciclib, temozolomide, and radiation in DIPG PDOX models: Insights from single-cell RNA-seq on cellular subtypes and genes critical for responsiveness and resistance.

2073 Background: Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, with two-year survival less than 10%. Radiation therapy (XRT) offers limited survival benefits, and effective therapies are urgently needed. This study investigates the FDA-approved CDK4/6 inhibitors-Abemaciclib, XRT, and temozolomide (TMZ) as a therapeutic regimen for DIPG using organoids and patient-derived orthotopic xenograft (PDOX) models, aiming to improve survival outcomes and gain insights into the underlying cellular and molecular mechanisms of DIPG treatment responsiveness and resistance to support rapid translation into clinical trials. Methods: The efficacy of Abemaciclib, XRT, TMZ, and their combinations was evaluated in DIPG organoids and PDOX models (IBs-A0317DIPG and IBs-9119DIPG, H3.3K27M mutation). In vitro, PDOX organoids were treated with Abemaciclib, TMZ, with/without XRT. Synergy was assessed using the Bliss Independence model. In vivo, six treatment arms were tested: (1) control, (2) XRT (2 Gy/day × 5), (3) Abemaciclib (75 mg/kg, p.o., 14 days), (4) Abemaciclib + XRT, (5) TMZ (50 mg/kg, p.o., 5 days) + XRT, (6) Abemaciclib + TMZ + XRT. Single-cell RNA sequencing and IHC were used to assess cellular subtypes responses, gene expression changes, and resistance mechanisms. Results: In DIPG organoids, the combination treatment yielded Over Bliss values > 0 (0.25 and 0.58 in A0317DIPG and 9119DIPG models, respectively) demonstrating synergistic activities. In PDOX models, the triple therapy showed improved median survival compared to other treatment arms and significant survival advantage over control ( p = 0.0157) and Abemaciclib alone ( p = 0.0461) in A0317DIPG, and control ( p < 0.0001), XRT alone ( p = 0.0032), Abemaciclib alone ( p = 0.0006), Abemaciclib + XRT ( p = 0.0046), and TMZ + XRT ( p = 0.0001) in 9119DIPG models. Single-cell RNA sequencing revealed six tumor subtypes: AC-like, NPC-like, OPC-like, MES-like, mitotic, and radiation-resistant cells. The triple therapy increased NPC-like and mitotic cell populations while decreasing AC-like and OPC-like cells in both models. Additionally, we identified a novel radiation-resistant subpopulation that expanded after XRT treatment. Dynamic gene expression analysis in different cell types identified key target genes and cell-type specific pathways that mediate therapy responsiveness and resistance. Conclusions: Our study demonstrates that the combination of Abemaciclib, TMZ, and XRT offers a novel, synergistic approach for DIPG, significantly improving survival in preclinical PDOX models. Single-cell RNA sequencing reveals the roles of different cell types and molecular changes underlying resistance, highlighting potential targets for future anti-resistance strategies in DIPG management.