Project Portugal 2030

Summary

Poor clinical responses and the rapid development of resistance hindered the approval of KRAS inhibitors for the treatment of metastatic CRC1,5. As 50% of metastatic CRCs exhibit a KRAS mutation6,7, it is estimated that nearly 0.5M individuals succumb annually due to the lack of personalized treatment8. This hints at a massive need for understanding the mechanisms CRC cells use to evade KRAS inhibition to develop better treatments and improve patients’ lives. Identification and targeting of genetic mechanisms of resistance in relapsed tumors9 is currently the better, though poorly efficient, alternative to delay the dismal outcome of disease progression upon treatment. Moreover, this approach entails that we are always one step behind tumor evolution. To act earlier and efficiently, it is crucial to understand the adaptations that occur between treatment initiation and relapse, which induce drug tolerance and allow tolerant cells to survive long enough to acquire (epi)genetic resistance mechanisms. With the OVERARCHING GOAL of improving CRC response to KRAS inhibitors, we will integrate longitudinal analyses of response to KRAS inhibition with lineage tracing and single-cell analysis of organoids and mouse models to delve into the role of chromatin reprogramming in tolerance and progression to resistance. By harnessing this in-depth knowledge, we plan to devise therapeutic strategies that prevent recurrence by blocking the path to resistance. Specifically, we aim to: 1. Establish a library of clones with distinct capacities to withstand KRAS inhibition. 2. Evaluate the dynamics of chromatin packing scaling and chromatin accessibility during evolution towards resistance. 3. Infer transcriptional trajectories and dissect the diversity within cell states. 4. Assess the transcriptional responsiveness of KRAS-inhibited cells to tumor microenvironment factors. 5. Devise therapeutic strategies to hinder the capacity of KRAS-inhibited cells to persist and resist. Aims 1-2 will harness our team's expertise in cancer biology, physical genomics, lineage tracing, and bioinformatics to explore the dynamics of chromatin structural changes and their effects on the transcriptional landscape of cells throughout treatment. Aim 3 will leverage the team's expertise in analyzing cancer cell-TME interactions. Through Imaging Mass Cytometry and in vitro experiments, we aim to identify specific TME niches and molecules that prime relapse and shape the features of tolerant clones worth exploiting therapeutically. Aim 4 builds on our prior data and KICk-CANCER’s data to put forward different strategies worth testing as potential treatments to synergize KRAS inhibition and enhance apoptosis, preclude the emergence of resistance clones, or reduce their heterogeneity to facilitate the action of further lines of treatment. Specifically, we will test a sequential combination of chemotherapy, KRAS/EGFR inhibition, and Digoxin (repurposing); target cell-intrinsic or TME-induced pathways underlying the survival of drug-tolerant persisters or resistant clones; explore the permissive responsive state of the chromatin structure of KRAS-inhibited cells as a unique opportunity to develop next-generation therapeutic strategies based on differentiation-restoring treatments to convert high malignant undifferentiated cancer cells into terminally differentiated cells of low tumorigenicity and higher sensitivity to apoptosis.