SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, with a one-year survival rate of only 20%. A major barrier to treatment is that activating mutations in KRAS (e.g. KRASG12D) drive >90% of PDAC, but there are currently no effective drugs that target this classic oncogene. PDAC is also characterized by the build- up of more compressive stress than any other tumor. The effects of mechanical compressive stress are also poorly understood. Compressive stress distorts and compresses cells leading to increased intracellular molecular crowding. This crowding has two key effects: molecular motion is inhibited, and also molecular assembly is increased as molecules are pushed together. We have found that increased molecular crowding greatly favors phase separation, and drives stress granule formation. Our original hypothesis was that KRASG12D oncogene activation would confer resistance to mechanical compression. In fact, the opposite was true: KRASG12D softens cells, leading to increased susceptibility to compressive stress. The consequent high molecular crowding increased stress granules, which we found actually help alleviate molecular crowding by sequestering RNA. We are now determining if stress granules are key for survival under compressive stress. High crowding and distortion also led to imbalances in the physical properties of mitotic spindles, issues with DNA replication, and frequent nuclear ruptures, all of which contribute to an extremely high rate of mitotic errors when KRASG12D and compression are combined. We therefore, investigated the new hypothesis that the combination of KRAS mutation and compression accelerate cancer cell evolution. In support of this idea, we found frequent and highly stereotyped aneuploidies in cells that we evolved in vitro under compression for one month. We also saw frequent whole genome duplications and mutation of TP53. The evolved cells were able to outcompete parent cells when grown under mechanical compression, indicating that they had adapted to this perturbed mechanical environment. We will now determine the mechanisms of this adaptation. We also identified Myc in a screen for genes that conferred resistance to compression. Myc is also present on chromosome 8, which we found was frequently gained in both our in vitro evolution experiments, and in patient PDAC biopsy samples. We hypothesize that genes and signaling pathways that are crucial for cancer cell survival under compression, will represent new therapeutic targets for solid tumors, especially highly mechanically perturbed tumors like PDAC. We are well positioned to discover these genes by analysis of large numbers of mechanically evolved clones, all of which originated from an isogenic parent. New approaches to treat PDAC are desperately needed. Therefore, the ultimate translation of our research has the potential for significant impact.