Rashi Raghulan

The CDK4/6 inhibitor palbociclib increases the antitumor activity of the RAS inhibitor in mouse models of PDAC. A, Scheme of experimental design (KPC1 orthotopic transplant into wild-type C57Bl/6 mice). B, Percentage change in tumor volume compared with day 0 as measured by weekly ultrasound. The y-axis indicates the percentage change in tumor volume, and the x-axis indicates the time point after treatment initiation. Each line represents the treatment group average. Statistical analysis: A two-tailed Mann–Whitney test was performed to compare the percentage change in tumor volume between the remaining RMC-7977-treated (n = 4) and RMC-7977 + palbociclib–treated (n = 5) mice at day 28. The P value is shown. C, Scheme of experimental design (KPC GEMM model). D, Percentage change in tumor volume compared with day 0 at 14 days after treatment initiation in KPC GEMMs following treatment with indicated agents. In mice that did not survive until day 14 ultrasound measurement, day 7 measurements are shown (indicated by a cross). Statistical testing: Ordinary one-way ANOVA, comparing the mean of each treatment group with the mean of every other treatment group and correcting for multiple comparisons with the Tukey test. Only statistically significant comparisons or relevant comparisons are shown. Sample size: vehicle (n = 6), palbociclib (n = 8), RMC-7977 (n = 7), RMC-7977 + palbociclib (n = 8). E, Probability of survival (%) in KPC GEMMs following treatment with indicated agents. Statistical testing: A log-rank (Mantel–Cox) test was performed to compare survival curves, and P values are shown. Sample size: vehicle (n = 6), palbociclib (n = 8), RMC-7977 (n = 7), RMC-7977 + palbociclib (n = 8). F, Percentage mean tumor volume change from day 0 at 21 days after treatment initiation in various CDX and PDX models. The mRECIST score was determined based on percentage mean tumor volume change, where mCR > 80% regression, mPR = 30%–80% regression, mSD = 30% regression – 30% growth, and mPD > 30% growth. Statistical testing: A two-way ANOVA with multiple comparisons, comparing the mean of each treatment group within each model shown and correcting for multiple comparisons with a Tukey test. Only daraxonrasib + abemaciclib (combo) versus abemaciclib or daraxonrasib in KP-4 was statistically significant (P < 0.0001 for both comparisons). The number of mice per treatment group per model is shown in G. G, Kaplan–Meier plot of progression-free survival in various CDX and PDX models, where progression is defined by tumor volume doubling from baseline. Statistical testing: A log-rank (Mantel–Cox) test was performed to compare survival curves, and P values are shown. Models are pooled, and the number of mice per treatment group is shown in the figure. (A and C, Created with BioRender.com.)

A triple combination consisting of RMC-7977, palbociclib, and a CD40 agonist maintains long-term tumor control despite the presence of residual disease. A, Scheme of experimental design (KPC1 orthotopic transplant into wild-type C57Bl/6 mice). B, Percentage change in tumor volume compared with day 0 as measured by weekly ultrasound. The y-axis indicates the percentage change in tumor volume, and the x-axis indicates the time point after treatment initiation. Each dot represents an individual mouse (n = 6 per treatment group at treatment initiation). Statistical testing: Two-way ANOVA with multiple comparisons (comparing each treatment group against every other treatment group within the same time point), with Tukey testing to correct for multiple comparisons. Only statistically significant comparisons are shown. At 28 days, n = 5 RMC-7977 + IgG mice remained, and n = 6 remained for all other treatment groups. At 35 days, n = 5 RMC-7977 + IgG mice and RMC-7977 + palbociclib + IgG mice remained, and n = 6 RMC-7977 + FGK4.5 mice and RMC-7977 + palbociclib + FGK4.5 mice remained. At 42 days, n = 4 RMC-7977 + IgG mice and RMC-7977 + palbociclib + IgG remained and n = 6 RMC-7977 + FGK4.5 mice and RMC-7977 + palbociclib + FGK4.5 mice remained. C, Representative snapshots of ultrasound imaging from RMC-7977 + palbociclib + IgG2b (top row) or RMC-7977 + palbociclib + FGK4.5 (bottom row) at indicated time points. Snapshots show the largest tumor cross-section. The red dashed line indicates tumor borders. D, Representative hematoxylin and eosin (H&E) images of residual tissue following 42 days of indicated treatment. The black dashed line surrounds residual tumor tissue. E, Scheme of experimental design (KPC1 orthotopic transplant into wild-type C57Bl/6 mice). F, Spaghetti plot showing percentage change in tumor volume compared with day 0 as measured by weekly ultrasound. The y-axis indicates the percentage change in tumor volume, and the x-axis indicates the time point after treatment initiation. Each line represents an individual mouse. G, Probability of survival (%) following treatment with indicated agents. RMC-7977 + palbociclib + IgG2b is not shown, as mice were euthanized prior to reaching humane endpoints. (A and E, Created with BioRender.com.)

CD40 agonist prolongs responses to RMC-7977 + palbociclib

CD4 T cells prevent outgrowth of residual disease. A, Scheme of experimental design (KPC1 orthotopic transplant into wild-type C57Bl/6 mice). B, Percentage change in tumor volume compared with day 0, as measured by weekly ultrasound. The y-axis indicates the percentage change in tumor volume, and the x-axis indicates the time point after treatment initiation. Each dot represents an individual mouse. Statistical testing: Two-way ANOVA with multiple comparisons (within each time point, comparing treatment groups with every other treatment group) and correcting for multiple comparisons with a Tukey test. All statistically significant comparisons are shown. C, Representative hematoxylin and eosin (H&E) images of residual tissue following 42 days of indicated treatment. D, Scheme of experimental design for E–H (KPC1-ZsGreen orthotopic transplant into wild-type C57Bl/6 mice). E, Fraction of CD8+ T cells out of total CD45 cells by flow cytometry at 7 days after the initiation of indicated treatments. See F for the legend. Statistical testing for E–G: Ordinary one-way ANOVA with multiple comparisons, comparing the means of each treatment group with every other treatment group and correcting for multiple comparisons using a Sidak test. Only relevant statistically significant comparisons are shown. F, Fraction of CD4+ T cells out of total CD45 cells by flow cytometry at 7 days after the initiation of indicated treatments. Each dot represents an individual mouse. G, Fraction of tumor cells (%) positive for MHC-II by flow cytometry at 7 days after the initiation of indicated treatments. See F for the legend. Each dot represents an individual mouse. Statistical testing: Ordinary one-way ANOVA, comparing the means of every treatment group with every other treatment group and correcting for multiple comparisons with a Sidak test. All statistically significant comparisons are shown. H, Fraction of CD4 T cells (%) positive for indicated cytotoxicity and activation of Th1 markers by flow cytometry 7 days after treatment initiation. Statistical testing: Two-way ANOVA with multiple comparisons, comparing the mean of each treatment group with every other treatment group within the relevant time point and correcting for multiple comparisons with a Tukey test. Only statistically significant comparisons are shown. I, Representative IF images of residual tissue following 42 days of indicated treatments (see scheme in A) staining for pan-CK, Ki67, cleaved caspase 3, and DAPI. (A and D, Created with BioRender.com.)

Samples collected for single cell RNA-sequencing studies

Palbociclib increases the anti-tumor activity of RMC-7977

Combined RAS and CDK4/6 inhibition drives tumor cells into a senescence-like state. A, Scheme of experimental design for B–D and Supplementary Fig S3A–S3I (KPC1-ZsGreen orthotopic transplant into wild-type C57Bl/6 mice). B, Representative images of pancreatic tumor tissues following detection of β-galactosidase activity using the chromogenic substrate X-Gal. C, Quantification of IF staining of pancreatic tumor tissues for β-galactosidase activity in mice 4 hours, 3, 7, or 14 days after treatment initiation. Each dot represents an individual mouse (average of 3–5 ∼120,000 µm2 regions per mouse). Where data from two separate experiments were available, mice from experiments were merged (n = 3–8 mice per treatment group). Statistical testing: Ordinary one-way ANOVA, comparing preselected pairs of columns (within treatment time point vs. vehicle only) and correcting for multiple comparisons with a Bonferroni test. Only statistically significant comparisons are shown. D, Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) showing tumor cells from each treatment group classified as “Sen-High” in blue and those classified as “Sen-Low” in yellow. “Sen-High” cells are defined as those that are positive for three senescence signatures in Supplementary Fig. S3F–S3H). Pie charts below show the fraction of total cells classified as Sen-High versus Sen-Low in each treatment group. The number of sequenced tumor cells is displayed for each treatment group. E, Scheme of experimental design (KPC1-ZsGreen-Watermelon orthotopic transplant into NSG mice) to trace the proliferative history of tumor cells following the indicated treatments. The strategy for gating DAPI−, ZsGreen+ tumor cells based on H2B-mCherry levels is shown. F, Fraction of tumor cells with highly retained (stably arrested since treatment initiation), mid, or low H2B-mCherry following the indicated treatments. Statistical testing: Two-way ANOVA, comparing the mean of each treatment group within each H2B-mCherry with every other treatment group. Multiple comparisons testing was corrected for with a Tukey test, and statistically significant results are reported here. H2B-mCherry high: vehicle versus RMC-7977 + palbociclib (P = 0.0458). H2B-mCherry mid: vehicle versus RMC-7977 + palbociclib (P < 0.0001); RMC-7977 versus RMC-7977 + palbociclib (P < 0.0001). H2B-mCherry low: vehicle versus RMC-7977 (P = 0.0128); vehicle versus RMC-7977 + palbociclib (P < 0.0001); RMC-7977 versus RMC-7977 + Palbociclib P < 0.0001). (A and E, Created with BioRender.com.)

Palbociclib does not increase RASi-driven cell death or affect RASi-driven phospho ERK inhibition

Characterizing RAS(ON) inhibitor-based combination therapies in a differentiated mouse model of PDAC

CD4 T-cell production of IFN-γ enables long-term tumor control. A, Expression of select cytotoxic and Th1-related genes in CD4 T cells from the scRNA-seq dataset. B, Expression of select genes encoding IFN-γ sensing machinery and IFN-γ–inducible genes in Sen-High versus Sen-Low cells. C, Scheme of experimental design (KPC1 orthotopic transplant into wild-type C57Bl/6 mice). D, Probability of survival (%) in mice following treatment with indicated agents. Statistical testing: A log-rank (Mantel–Cox) test was performed to compare survival curves, and P values are shown. E, Scheme of experimental design (KPC1 orthotopic transplant into wild-type C57Bl/6 mice). F, Representative images of pancreatic tumor tissues following detection of β-galactosidase activity using the chromogenic substrate X-Gal. G, Quantification of the fraction of SA-β-gal positive area out of total area (%). Each dot represents an individual mouse (n = 3–5 per treatment group), and the value shown is the average of 3 ∼159,000 µm2 regions per mouse tumor tissue (with the exception of two mice, where tumor tissue was only large enough to quantify 1–2 regions). Statistical testing: One-way ANOVA, comparing the mean of every column with the mean of every column within that time point and correcting for multiple comparisons with a Holm–Sidak test. All comparisons are shown. (C and E, Created with BioRender.com.)