A Sequential Targeting Strategy Interrupts AKT-Driven Subclone-Mediated Progression in Glioblastoma

PURPOSE: Therapy resistance and fatal disease progression in glioblastoma are thought to result from the dynamics of intra-tumor heterogeneity. This study aimed at identifying and molecularly targeting tumor cells that can survive, adapt, and subclonally expand under primary therapy. EXPERIMENTAL DESIGN: To identify candidate markers and to experimentally access dynamics of subclonal progression in glioblastoma, we established a discovery cohort of paired vital cell samples obtained before and after primary therapy. We further used two independent validation cohorts of paired clinical tissues to test our findings. Follow-up preclinical treatment strategies were evaluated in patient-derived xenografts. RESULTS: We describe, in clinical samples, an archetype of rare ALDH1A1+ tumor cells that enrich and acquire AKT-mediated drug resistance in response to standard-of-care temozolomide (TMZ). Importantly, we observe that drug resistance of ALDH1A1+ cells is not intrinsic, but rather an adaptive mechanism emerging exclusively after TMZ treatment. In patient cells and xenograft models of disease, we recapitulate the enrichment of ALDH1A1+ cells under the influence of TMZ. We demonstrate that their subclonal progression is AKT-driven and can be interfered with by well-timed sequential rather than simultaneous antitumor combination strategy. CONCLUSIONS: Drug-resistant ALDH1A1+/pAKT+ subclones accumulate in patient tissues upon adaptation to TMZ therapy. These subclones may therefore represent a dynamic target in glioblastoma. Our study proposes the combination of TMZ and AKT inhibitors in a sequential treatment schedule as a rationale for future clinical investigation.