Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-Mutant Pancreatic Cancer

Abstract

Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK-MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K-AKT-mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor-based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC.

Significance: CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer.

Figure 1.

CDK4/6 inhibition reduces PDAC cell viability and induces compensatory signaling changes. A, Representative western blots for indicated proteins after 72 hours treatment with siRNA targeting CDK4, CDK6, the combination, or non-silencing control (NS). Protein expression ratio to NS indicated. B, Cell proliferation after siRNA-based silencing of protein expression in indicated cell lines. Cell proliferation was measured by automated direct cell counting in a 96-well format 5 days post-knockdown (n ≥ 3, error bars SD). C, Average EC₅₀ (nM) for palbociclib in the indicated PDAC cell lines in an anchorage-dependent proliferation assay. D, Top 25 largest fold increases (red) or decreases (blue) in protein phosphorylation or expression compared to vehicle control upon treatment with 200 nM palbociclib for 5 days, as measured using RPPA in six cell lines (full dataset in Table S1). E, Dot-plot of values for each cell line from panel D for indicated proteins and pathways. Activating ERK phosphorylation at T202/204 was the largest upregulation in response to CDK4/6i treatment in the dataset. F, Microscopy images (40x) of Pa16C cells constitutively expressing the mCherry-ERK-KTR treated with 400 nM palbociclib or DMSO. Higher ERK activity causes nuclear export of the ERK-KTR reporter, thus leading to higher cytoplasmic/nuclear fluorescence ratio. Scale bar = 60 μm. G, Palbociclib causes significantly higher ERK activity compared to vehicle. ERK inhibitor SCH772984 at 200 and 1000 nM shown as a positive control (error bars SEM).

Figure 2

CDK4/6 inhibitors synergize with ERK inhibitors to reduce viability and overcome CDK4/6 inhibitor-mediated signaling compensatory changes. A, Cell viability dose-response curves for ERK inhibitor SCH772984 in combination with fixed indicated concentrations of palbociclib under anchorage-dependent cell culture conditions (representative of at least three independent experiments). B, Three-dimensional representation of (A), color-coded with BLISS synergy scores (red = synergy, blue = antagonism). C, Maximum BLISS synergy scores across a panel of PDAC cell lines (BLISS <1 is synergistic). D, Fourteen-day colony formation assays for the palbociclib and SCH772984 combination (representative of two independent experiments). E, Western blot of cells treated with palbociclib alone or combined with SCH772984 after three days treatment (representative of two independent experiments). pRB expression ratio to Tubulin is indicated. F, Western blot for RB phosphorylation with one to five days treatment of palbociclib, SCH772984, or the combination over a wide dosing range (representative of two independent experiments).

Figure 3.

Combination of CDK4/6i and ERKi alters cell cycle regulation, increases apoptosis, and induces compensatory cellular signaling. A, Flow cytometry-based annexin-V/PI staining for apoptosis in MIA PaCa-2 cells induced by CDK4/6i palbociclib (600 nM), ERKi SCH772984 (500 nM), or the combination after five days of treatment (representative images of three independent experiments). B, Quantified percent apoptosis after palbociclib, SCH772984, or the combination at two palbociclib concentrations across a PDAC panel (error bars, SD of at least two independent experiments) from flow cytometry analyses (panel A). C, Log2 fold-change RPPA results after treatment with CDK4/6i, ERKi, or the combination for 14 days across six PDAC lines. Shown are the top and bottom 20% quantile (upregulation, red or downregulation, blue) to combination treatment. D, Log₂ fold-change (FC) for RB (S780) phosphorylation and E, MYC expression upon CDK4/6i, ERKi, or combination treatment over a time course of one to 14 days.

Figure 4.

Combined CDK4/6i and ERKi inhibition induces apoptosis in PDAC organoids. A, PDAC patient-derived hT2 organoids treated with indicated concentrations of palbociclib and ERK inhibitor SCH772984 imaged at 4x resolution (representative images of three independent experiments, scale bar = 500μm). B, Organoid (hT2 and hM1a) viability as measured by CellTiter-Glo (Promega) treated with SCH772984 and palbociclib in a dose-response matrix (representative of three independent experiments). C, Three-dimensional representation of the luminescence (viability) scores from (A), colored by BLISS synergy (red) or antagonism (blue) between CDK4/6i and ERKi in hT2 and hM1a organoids. D, Induction of caspase activity as measured by Caspase-Glo (Promega) in organoid models treated with palbociclib and SCH772984 (representative of two independent experiments).

Figure 5.

Loss-of-function CRISPR-Cas9 screen discovers CDK4/6i-sensitizing genes. A, Composition of a loss-of-function CRISPR “druggable genome” library targeting 2,240 genes. B, Screening workflow: cells were infected with lentivirus expressing Cas9 and barcoded sgRNA constructs in a pooled format, with an initial sample collected after transfection to measure library representation (T0). Cells were grown for an additional seven days to for DNA alteration and initial selection of early essential genes (sample T7 collected). Cells were then split into two replicates of palbociclib- or control-treatment arms and dosed continually (200 nM) for two or four weeks. C, Average difference beta score across all six cell lines and both timepoints. Negative values mean loss of indicated protein enhances CDK4/6 inhibitor palbociclib sensitivity. D, Top 50 genes with the most palbociclib-selective difference beta scores by median across all cell lines and timepoints using MAGeCK-MLE. E, Average RSA sensitivity LogP score across all six cell lines and both timepoints. Negative values mean loss of indicated proteins enhance palbociclib sensitivity.

Figure 6.

Palbociclib-sensitizing CRISPR hits validated using siRNA counterscreen and small-molecule inhibitors. A, Indicated gene hits from the CRISPR screen were individually silenced using siRNA, then treated with palbociclib. Shown is the fold-shift in the EC₅₀ of palbociclib compared to cells treated with non-specific siRNA (red = more sensitive, blue = less sensitive). Data is shown as average of three independent replicates. B, Example of data used to generate (A). Dose-response curves were measured for palbociclib in siRNA-treated cells, and cell viability was measured using direct cell counting. BLISS synergy score was calculated for each combination (red = synergy). C, Indicated compounds were tested against the panel of PDAC lines used in the CRISPR screen in a dose-response matrix with palbociclib; on average, drugs were synergistic (red), antagonistic (blue), or additive (white). D-E, Example matrix dose-response curves for SRC family inhibitor bosutinib (Pa02C) and HDAC inhibitor quisinostat (Pa16C) at different fixed concentrations of CDK4/6 inhibitor palbociclib (upper panel), with BLISS synergy shown mapped on the 3D dose-response matrix (lower panel).

Figure 7.

CDK2 inhibition synergizes with CDK4/6i by cooperatively inhibiting cell cycle proteins and MYC. A, Violin plot of cell cycle distribution for the six PDAC cell lines used in the CRISPR screen and treated with DMSO, palbociclib, or CDK2/4/6 inhibitor PF-06873600 (each at 300 nM) (representative of three independent experiments). B, Percent apoptotic cells after treatment with palbociclib or CDK2/4/6 inhibitor PF-06873600 (200 nM) for five days (error bars, SD of at least three independent experiments). C, RPPA results after 14 days PF-06873600 treatment showing increased (red, log2 fold-change) or decreased protein (blue). D-E, Change in indicated proteins over one, three, and 14 days with PF-06873600. F, ERK activation by PF-06873600 as measured using the ERK-KTR reporter readout. G, Viability dose-response curves for response matrix in PDAC-derived organoids treated with PF-06873600 plus ERKi for five days (representative of three independent experiments).