Supplementary Table 1. Flow cytometry antibody details:

Supplementary Figure 9. Intermittent CKI27 treatment and GITR engagement relieves suppressive effects of MEK inhibition on T cell proliferation, cytokine production, and effector function. (A-C) Human PBMCs were labelled with CTV, sub-optimally stimulated with 1:25 or 1:100 CD3/CD28 Dynabeads, and treated with DMSO, continuous CKI27 (96hr), washout CKI27 (24hr on, 72hr off), and/or GITR-L; n=2-3. (A) % proliferation of CTVlow CD8+ and CD4+ T cells. (B) FACS analysis of co-inhibitory, co-stimulatory, and activation markers on CD8+ and CD4+ T cells. Heatmaps represent fold changes of positive percentages of each marker normalized to DMSO. (C) Cytokine analysis of supernatants collected from all PBMCs. Heatmap represents fold changes of concentrations (pg/mL) of proteins normalized to DMSO. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 2. MEK inhibition with CKI27 increases MHC and checkpoint ligand expression. Murine tumor cell lines were treated with DMSO or CKI27 for 72 hr and either with or without IFNγ (5ng/mL) for the last 24hr; n=3. FACS analysis of representative histograms for the MFI of MHC-I (H2Kb/Kd and H2Db/Dd), MHC-II, PD-L1, CD80 and CD86 are shown.

Supplementary Figure 13. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while destabilizing Tregs in LLC tumor. (A) Schema of LLC tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Numbers of cells/mg of TILs; n=4-5. (C) Phenotypes of TILs; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 12. The triple combination increases activation of CD8+ T cells, CD4+ Teffs, and Tregs in CT26 TDLN. (A) Schema of CT26 tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Absolute number of immune cell populations in the TDLN; n=4-5. (C) Phenotypes of T cells from the TDLN; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplemental Figure 11. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while Tregs remain unaffected in LLC TDLN. (A) Schema of LLC tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Image of TDLNs from LLC tumor bearing mice. (C) Gating strategy for all in vivo flow experiments. (D) Absolute number of immune cell populations in the TDLN; n=4-5. (E) Phenotypes of T cells from the TDLN; n=9-10. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Table S5. Preliminary mutation analysis

Supplementary Figure 14. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while destabilizing Tregs in CT26 tumor. (A) Schema of CT26 tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Tumor weights, numbers of cells/mg, and CD8:Treg ratio of TILs; n=4-5. (C) Phenotypes of TILs; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 5. Intermittent CKI27 allows for immune cell recovery in the spleen, increases frequencies in the TDLN, and inhibits TILs similarly to continuous treatment. (A) Schema of LLC tumor bearing mice treated with vehicle, daily 2mg/kg CKI27, or intermittent 5mg/kg 4on/3off CKI27. Mice were treated in a staggered schedule and all timepoints were harvested on day 23. (B-D) All fold changes were calculated by normalizing to DMSO. (B) Fold changes of absolute number (cells/uL) of spleen immune cell populations. (C) Fold changes of absolute number (cells/uL) of TDLN immune cell populations. (D) Fold changes of tumor weights (mg) and TILs (cells/mg); n=4-5. Data are shown as mean±SEM. Unpaired, nonparametric Mann-Whitney test was used to compare each time point. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 1. MEK inhibition with CKI27 increases MHC and checkpoint ligand expression. (A-B) Murine tumor cell lines were treated with DMSO or CKI27 for 72 hr and either with or without IFNγ (5ng/mL) for the last 24hr; n=3. FACS analysis of (A) MHC-I (H2Kb/Kd and H2Db/Dd) and MHC-II and (B) PD-L1, CD80 and CD86 surface expression. Median fluorescence intensity (MFI) values were normalized to DMSO and log transformed. Data are shown as mean±SEM.

Supplementary Figure 7. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of co-stimulatory markers. Representative dot plot data for FACS analysis of co-stimulatory markers expressed by CD8+ T cells.

Supplementary Figure 15. The triple combination favorably alters the genetic profile of immune cells in the TDLN. LLC tumor bearing mice were treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. TLDNs were harvested on day 21 (7 days post treatment). Live CD45+ cells were FACS sorted and processed for sc-RNA sequencing. (A-B) Heatmap showing top genes expressed by each cluster for (A) CD8+ and (B) CD4+ T cells. (C) UMAPs of each treatment groups showing different clusters and annotations. (D) UMAPs of CD8+ and CD4+ T cells from each treatment group showing specific activation genes and proteins.

Supplementary Figure 10. The triple combination reduces tumor growth, is T cell dependent, and protects from re-challenge in LLC and CT26. (A-D) LLC tumor bearing mice were treated with vehicle, isotypes, GITR, CTLA-4, 5mg/kg 4on/3off CKI27, and/or CD8 for 4 weeks and tumor growth was monitored over time. (A) Average tumor growth (volume, mm3) of immunocompetent mice. (B) Average tumor growth (volume, mm3) of immunodeficient mice. (C) Average tumor growth (volume, mm3) of CD8 depleted mice. (D) Average tumor growth (volume, mm3) of mice from (A) that were re-challenged. (E-H) CT26 tumor bearing mice were treated with vehicle, isotypes, GITR, αCTLA-4, 2mg/kg 4on/3off CKI27, and/or αCD8 for 4 weeks and tumor growth was monitored over time. (E) Average tumor growth (volume, mm3) of immunocompetent mice. (F) Average tumor growth (volume, mm3) of immunodeficient mice. (G) Average tumor growth (volume, mm3) of CD8 depleted mice. (H) Average tumor growth (volume, mm3) of mice from (E) that were re-challenged. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 8. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of activation markers. Representative dot plot data for FACS analysis of activation markers expressed by CD8+ T cells.

Supplementary Figure 4. Intermittent CKI27 treatment partially relieves suppressive effects of MEK inhibition on T cell proliferation, cytokine production, and effector function. (A-C) Human PBMCs were labelled with CTV, sub-optimally stimulated with 1:25 or 1:100 CD3/CD28 Dynabeads, and treated with DMSO, continuous CKI27 (96hr) or washout CKI27 (24hr on, 72hr off); n=2-3. (A) Proliferation fold change of CTVlow CD8+ and CD4+ T cells was calculated by normalizing to DMSO. (B) FACS analysis of co-inhibitory, co-stimulatory, and activation markers on CD8+ and CD4+ T cells. Heatmaps represent fold changes of positive percentages of each marker normalized to DMSO. (C) Cytokine analysis of supernatants collected from all PBMCs. Heatmap represents fold changes of concentrations (pg/mL) of proteins normalized to DMSO. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 3. MEK inhibition with CKI27 increases HLA and checkpoint ligand expression. (A-B) Human tumor cell lines were treated with DMSO or CKI27 for 72hr and either with or without IFNγ (10ng/mL) for the last 24hr; n=3. FACS analysis of (A) HLA-ABC and HLA-DR and (B) PD-L1, CD80 and CD86 surface expression. MFI values are shown as mean±SEM.

Supplementary Figure 6. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of co-inhibitory markers. (A) Gating strategy for all T cell activation assays. (B) Representative dot plot data for FACS analysis of co-inhibitory markers expressed by CD8+ T cells.

Supplementary Table 2. Gene list associated with each cluster.

Peritoneal carcinomatosis (PC) is a common yet deadly manifestation of gastrointestinal cancers, with few effective treatments. To identify targetable determinants of peritoneal metastasis, we focused on appendiceal adenocarcinoma (AC), which metastasizes almost exclusively to the peritoneum. No stable preclinical models of AC exist, limiting drug discovery and representing an unmet clinical need. We establish a stable biobank of 16 long-term cultured AC patient-derived tumor organoids (PDTOs). We establish an organoid orthotopic intraperitoneal xenograft model that recapitulates diffuse PC and show that PC organoids retain increased metastatic capacity, decreased growth-factor dependency, and decreased sensitivity to standard-of-care chemotherapy relative to matched primary AC organoids. Single-cell profiling reveals dedifferentiation from differentiated states in primary AC into intestinal stem cell and fetal progenitor states in AC-PC, with upregulation of oncogenic signaling pathways. We identify KRASMULTI-ON inhibitor RMC-7977 and the Wnt-targeting tyrosine kinase inhibitor WNTinib as clinically actionable strategies to target AC-PC more effectively.

Inactivating mutations in Neurofibromin 1 (NF1), a RAS-GTPase, have been identified in a significant subset of melanomas, both concurrent with and exclusive of oncogenic BRAF and RAS mutations. However, FDA-approved therapies targeting the MAPK pathway have been largely inactive in this molecular cohort. To address the underlying biology, we first leveraged sequencing data from 1, 912 melanoma tumor/normal pairs generated as part of the MSK-IMPACT sequencing initiative and identified oncogenic mutations in TP53 as the most significant event common to NF1-inactivated melanoma, with oncogenic BRAF mutations frequently co-occurrent. Given the lack of models for studying NF1 loss, we next generated a cohort of genetically engineered mice harboring conditional alleles for Nf1fl/fl, Trp53fl/fl, and/or oncogenic BRAF V600E (BrafCA) and the transgene Tyr::CreERT2, restricting induction to melanocytes. While melanocyte-specific, homozygous knockout of Nf1 induced hyperpigmentation, it was insufficient for melanomagenesis. Combined Nf1/Trp53 knockout induced tumor formation with an incidence of 55.56%; tumor onset and hyperpigmentation were hastened upon the addition of oncogenic BRAF V600E. Other genetic cohorts induced tumors with variable penetrance and latency (BrafCA/Nf1, BrafCA/Trp53). All tumors displayed features of human melanoma including pigmented melanocytic and amelanotic spindle-shaped neoplasms, with most staining positive for S100. To facilitate preclinical and functional studies, we generated 22 mouse tumor-derived, syngeneic cell lines. As anticipated, loss of Nf1 conditioned the response to treatment with a BRAF monomer inhibitor (vemurafenib), while Nf1-inactivated cells retained sensitivity to MEK inhibition (trametinib) or the combination. Combined inhibition of BRAF/SHP2 inhibited ERK phosphorylation and cell proliferation in BrafCA/Nf1/Trp53-mutant cells; however, inhibition of ERK signaling was transient. Combination treatments that resulted in more durable inhibition of ERK, including the triple combination of RAF/MEK/SHP2 inhibitors, achieved tumor regression in mice with established BrafCA/Nf1/Trp53-null tumors as well as in a genetically similar human PDX of melanoma (SK-MEL-1273A). Overall, we find that addition of SHP2 to MEK or RAF inhibitors resulted in more durable MAPK pathway inhibition and greater tumor regression in mice bearing NF1-null tumors. The testing of additional combinations of novel agents that selectively target the MAPK pathway and other parallel pathways contributing to resistance and survival are ongoing. Olvania-Danyca Hilaire, Alexis M. Jones, Moriah H. Nissan, Sebastien Monette, Jordan Eichholz, Sandra Misale, Isabell Schulze, Naresh Vasani, Cailian Liu, Xia Yang, Elisa De Stanchina, Nikolaus Schultz, Michael F. Berger, Neal Rosen, Taha Merghoub, David B. Solit, Aphrothiti J. Hanrahan. Durable inhibition of ERK phosphorylation is required for tumor regression in novel human and mouse models of NF1-inactivated melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3935.