Thomas McFall

Figure S1. Role of KRASMUT-SOS axis in MAPK signaling

Figure S2. Ex-vivo organotypic slice culture methodology

Figure S3. RAS inhibitor resistance

Clinical-grade RAS inhibitors raise an unresolved question about whether KRAS alleles impose constraints on adaptive resistance that can be exploited therapeutically. Using daraxonrasib (RMC-6236), a multiselective RAS(ON) inhibitor, we compared resistance mechanisms between KRASG12D and KRASG12R, mutants with fundamentally different RAS network dynamics. Daraxonrasib inhibited mutant KRAS primarily through steric occlusion of effector binding while engaging wild-type RAS (RASWT) only modestly (∼20%). KRASG12R was marked by its inability to transactivate RASWT, and daraxonrasib-resistant KRASG12R pancreatic ductal adenocarcinoma (PDAC) cells utilized EGFR/RASWT-GTP signaling as the dominant adaptive route. In contrast, KRASG12D resistance arose through retained KRASG12D-GTP signaling, with a decrease in cyclophilin A (CypA) protein, the binding partner required for daraxonrasib activity. The shift from KRASG12R dependence to EGFR/RASWT dependence conferred sensitivity to trametinib. As clinical confirmation, a patient with KRASG12R PDAC who progressed after 10 months on daraxonrasib showed intratumoral EGFR/RASWT activation, and rapid 3D-bioprinted patient-derived tumoroid modeling predicted sensitivity to trametinib-based combination therapy. Despite the aggressive disease trajectory and lack of response to the two immediately preceding lines of therapy, sixth-line trametinib-based combination therapy achieved approximately 5 months of disease control, and this patient ultimately achieved 40 months of overall survival, far exceeding the 8 to 12 months median for metastatic PDAC. Collectively, these data establish a framework in which allele-specific RAS network topology dictates the adaptive resistance landscape, enabling rational selection of targeted therapies with meaningful clinical benefit in metastatic PDAC. Significance:Daraxonrasib resistance emerges through allele-specific routes, with CypA being downregulated in KRASG12D and reliance on EGFR/RASWT in KRASG12R cancer cells, informing mechanism-based therapeutic strategies for patients who progress on RAS inhibitors.

Figure S5. Characterization of toroid integrity by orthogonal methods

Figure S4 Patient Tumor Bioprinting

Figure S6. Sensitivity to trametinib

Pancreatic ductal adenocarcinoma (PDAC) is predominantly driven by oncogenic KRAS signaling and sustained by a desmoplastic, immunosuppressive microenvironment limiting therapeutic response. CD73-mediated adenosine signaling supports this immune-excluded, tumor-supportive microenvironment. Whether dual targeting of KRAS-MAPK and CD73 signaling creates a therapeutic opportunity to improve tumor control remains unexplored. Analysis of KRAS -mutant cancer cell lines revealed increased expression of NT5E and adenosine receptor ADORA2B . Pharmacologic and genetic suppression of oncogenic KRAS inhibited CD73 expression through a MAPK-dependent program. In syngeneic KPC tumors, conversely, CD73 inhibition with quemliclustat increased ERK phosphorylation in vivo , suggesting adaptive MAPK activation following CD73 blockade. Combined CD73 and RAS(ON) inhibition in vivo produced the strongest antitumor response, reducing endpoint tumor volume beyond either monotherapy. This combination significantly suppressed tumor proliferation and αSMA-positive stroma and resulted in a substantial reduction in CD163-positive macrophage infiltration. RAS-MAPK targeted therapy was also associated with increased CD8α-positive T cell infiltration and granzyme B-positive staining. As therapeutic responses in PDAC have been linked to the gut microbiome, we further examined treatment-associated changes in the microbiome. RAS-MAPK inhibition produced the most pronounced microbial alterations, which correlated with tumor volume and immune markers. Together, these findings identify KRAS–MAPK driven CD73 signaling as a therapeutic vulnerability in PDAC and support combined CD73 and RAS(ON) inhibition as a strategy to suppress tumor growth, remodel stroma, and alleviate tumor-associated immune suppression.

Metastatic pancreatic ductal adenocarcinoma (PDAC) has historically been managed with systemic therapy, and local therapies have, in general, been avoided in the absence of symptoms directly related to a specific metastasis. Even if a specific site of disease, such as a local recurrence, had the potential to cause symptoms in the future, there was a bias against prophylactic palliation. It was well-established that most patients die of systemic progression. This approach is now under pressure from four directions. Retrospective case series have reported survival durations longer than anticipated in highly selected patients with low-volume, single-organ disease. A randomized phase II trial reported that metastasis-directed therapy prolonged progression-free survival. Biologic data indicate that the site of metastatic relapse is encoded in gene expression profiles already present in the primary tumor. Second-line systemic therapy has become substantially more effective, which lengthens the interval over which control of an untreated metastasis may become clinically important. We trace the oligometastatic concept from the 1995 Hellman and Weichselbaum hypothesis to the 2026 OligoPanc consensus definition of up to three lesions confined to a single organ, examine the criteria proposed for patient selection, summarize the surgical and ablative radiotherapy literature, and consider the trials that will determine whether this is a treatable disease state or a favorable prognostic phenotype. We conclude that induction systemic therapy, followed by response assessment that weighs the absence of disease progression, Carbohydrate Antigen 19-9 (CA19-9) kinetics, and performance status, is a defensible route to local therapy directed at the metastases and the primary tumor, and warrants prospective study.