Objective Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4.
Design Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens).
Results Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach.
Conclusion This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.
- pancreatic cancer
- extracellular matrix
- molecular mechanisms
- cell cycle
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.
Significance of this study
What is already known on this subject?
Despite significant recent efforts into developing novel therapeutics, chemotherapy remains the standard of care for patients with advanced pancreatic cancer, despite only modestly improving overall survival.
We and others have previously shown that pancreatic cancer is a molecularly varied disease and requires development of personalised treatment strategies to improve survival rates.
Pancreatic cancer harbours frequent deregulation of the p16-cyclin D-CDK4/6-retinoblastoma (RB) protein pathway (CDK4 pathway), which could be therapeutically exploited.
What are the new findings?
Using a range of robust patient-derived xenograft and cell line models, as ‘avatars of matched patient pancreatic tumours’, we show exquisite sensitivity to CDK4/6 inhibitor combinations over standard therapy in subtypes of pancreatic cancer, associated with increased apoptosis, quiescence and reduced expression of markers associated with pancreatic stellate cell activity.
PD-0332991 facilitates remodelling of the extracellular matrix (ECM) to improve therapeutic efficacy of gemcitabine (GEM) in a subtype-specific manner and at multiple stages of disease progression: primary tumour growth, recurrence (second-line therapy) and the metastatic setting and may potentially be guided by a simple, clinically applicable biomarker (RB protein).
CDK4 inhibitor, PD-0332991 (palbociclib) hinders metastatic colonisation in the liver while sensitising cells to shear stress, commonly experienced by metastasising circulating tumour cells, an additional way in which CDK4 inhibition can directly impinge on metastasis in RB-high pancreatic ductal adenocarcinoma (PDA).
RB protein expression was identified as an independent prognostic factor for patient survival in two independent patient cohorts of pancreatic cancer and can be detected in a significant proportion of metastatic cases (67%). RB expression correlated between primary and matched metastatic human tissue.
CDK4 pathway inhibition alone or in combination with chemotherapy leads to multifaceted global inhibitory effects on tumour cells at different stages of PDA progression and reduction in key components within the tumour microenvironment (ECM), which collectively drive cancer cell proliferation, invasion and chemoresistance.
How might it impact on clinical practice in the foreseeable future?
Our findings, together with the recent clinical evidence on the efficacy of PD-0332991 in the treatment of breast cancer, support the further clinical companion biomarker-driven development of PD-0332991 as pancreatic cancer therapy, particularly in combination with chemotherapeutic, GEM.
The use of a tissue-based assay for RB expression could be adapted for clinical application in diagnostics. As high RB expression was detected in a significant proportion of patients with pancreatic cancer (~65%,~43% and 67% in cohorts of primary operable and metastatic PDA examined), it may enable stratification and enrichment for the potential responders to the CDK4/6 combination therapy.
These findings are timely and may be complementary and of direct relevance to the current non-biomarker-driven clinical trials testing PD-0332991 in pancreatic and other solid cancers (NCT02501902, NCT03065062).
Pancreatic ductal adenocarcinoma (PDA) is a molecularly diverse disease, driven by aberrations in at least 10 core signalling pathways.1–4 In addition to revealing new biological insight into the molecular pathogenesis of PDA, these studies have created significant opportunities to develop novel, tailored treatments, as we have recently shown.5 6 The standard of care chemotherapeutic gemcitabine (GEM),7 in combination with nanoparticle albumin-bound paclitaxel (nab-paclitaxel),8 or FOLFIRINOX (oxaliplatin, irinotecan, fluorouracil and leucovorin)9 offers only modest increases in patient survival in unselected populations.10 Selective measures to personalise treatment in this aggressive disease are therefore urgently required.
The p16-cyclin D-CDK4/6-retinoblastoma (RB) protein pathway (CDK4 pathway) promotes G1/S-phase transition and is one of the key signalling pathways deregulated in PDA.1 2 11 Cyclin D complexes with cyclin-dependent kinases (CDKs) 4 and 6, driving RB protein phosphorylation and G1 phase progression.12 In cancer, CDK4 activity and subsequent uncontrolled proliferation can be enhanced by homozygous deletion of CDKN2A gene (which encodes p16INK4A protein), CDK4/6 mutations and/or cyclin D1 (CCND1) overexpression or amplification.13
Discovery of cyclin D/CDK4 as an oncogene has fuelled development of small-molecule CDK inhibitors (CDKi). Selective CDK4/6i, including PD-0332991 (palbociclib), are showing great potential in the treatment of ER+/HER2– metastatic breast cancer.14–16 Although no biomarker of treatment response has been found, a key requirement for sensitivity to CDK4/6i in breast cancer may be linked to ER– positivity and dependence of the ER+ subtype on cyclin D1 signalling, enhancing CDK4/6 activity to drive proliferation.12 17
In PDA, short-term antiproliferative activity of CDK4/6i has been described.18 Here, we investigated the therapeutic efficacy of CDK4/6i and clinically applicable combinations with long-term follow-up in realistic advanced preclinical patient-derived and genetically engineered models of PDA. We reveal a new role of CDK4 inhibition in regulating primary tumour growth and stromal desmoplasia in several independent mouse models of RB-high PDA, impairing colonisation of secondary sites (liver) and resistance to shear stress, frequently experienced by circulating tumour cells (CTCs), while delaying disease progression in metastatic RB-high PDA. Detailed analyses reveal a complex mechanism of action with PD-0332991 exerting dual effects on both tumour cells and the surrounding environment. Assessment of RB expression, candidate CDK4/6i companion biomarker, revealed high prevalence in primary operable and metastatic human PDA. Collectively, these preclinical findings may offer promising new opportunities in subtype-specific targeting of primary and metastatic PDA.
Our Kinghorn Cancer Centre (TKCC) patient-derived xenografts (PDXs; n=45) and patient-derived cell lines (PDCLs; n=19) have previously been whole genome sequenced.1 PDCLs were confirmed by short tandem repeat (STR) profiling as unique (cellbankaustralia.com). Culture conditions, fluorescent ubiquitination-based cell cycle indicator (FUCCI) and luciferase introduction into PDCLs and associated in vitro studies: cell cycle, migration, invasion, shear stress, contraction and apoptosis assays are detailed in online supplementary information 2.
Survival studies were performed by subcutaneously implanting early third-passage PDX pieces (~4 mm3) into immunocompromised Balb/c-Fox1nuAusb (Nude) mice. PD-0332991, GEM and nab-Paclitaxel dosages were based on published studies,5 18 19 with treatment schedules, response measurement and setup for intrasplenic and orthotopic studies detailed in online supplementary information 2.
Patient materials and immunohistochemistry
Patient samples and data were acquired through the Australian Pancreatic Genome Initiative (APGI)/International Cancer Genome Consortium (ICGC) (n=200), New South Wales Pancreatic Cancer Network (NSWPCN; n=365) and the Royal North Shore Hospital metastatic cohort (RNSH; n=54), as described.1 5 20 Further cohort description and immunohistochemical procedures are outlined in online supplementary information 2.
Statistical analyses were performed using GraphPad Prism (V7.0.1, GraphPad, La Jolla, California, USA) and Statview (V5.0, SAS Institute Inc., Cary, NC, USA). Correlation analyses were performed using Pearson correlation coefficient test. Comparison between clinicopathological correlates was performed using Fisher’s exact test, Student’s t-test and Χ2 test, where appropriate. Survival analyses were performed using the log-rank test. For multiple treatment group comparisons, significance was determined by one-way analysis of variance, followed by Tukey post hoc multiple comparisons test where *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Clinicopathological variables shown by univariate analysis to have p<0.25 were entered into a Cox proportional hazards regression model for multivariate analysis. To assess the agreement of RB status between primary and metastatic PDAs, Cohen’s Kappa and 95% CIs were reported.
PD-0332991 sensitivity of pancreatic cancer patient-derived cell lines (PDCLs) correlates with high levels of total and phosphorylated RB protein
To systematically examine the efficacy of CDK4/6i PD-0332991 in PDA, we determined its toxicity in 19 whole genome-sequenced5 primary PDCLs and compared these data with several clinically used chemotherapeutics (see online supplementary table 1). PDCLs exhibited a broad range of PD-0332991 sensitivity (inhibitory concentration 50 (IC50) range 8 nM–35 µM), with a subset (26%) being highly sensitive (IC50 <1 µM). The ‘Genomics of Drug Sensitivity in Cancer’ (Wellcome Trust) database analysis revealed an association between RB1 mutations and pan-cancer PD-0332991 resistance (p=3.3×10–17, see online supplementary figure 1A). As RB1 mutations in pancreatic cancer are rare1 (1/19 PDCLs; see online supplementary table 1), we next correlated the baseline protein expression of key G1/S checkpoint components (see online supplementary figure 1B, supplementary table 2) with in vitro PD-0332991 response. Of note, total and phosphorylated RB (pRB; Ser780) levels were the most significant biomarkers predicting PD-0332991 sensitivity (figure 1A; r=−0.751; p=0.0002 and r=−0.728, p=0.0004, respectively), with no correlation between G1/S checkpoint mutations/expression and response to PD-0332991 or chemotherapeutics in pancreatic PDCLs (see online supplementary table 3, supplementary figures 1C–E).
Effective target modulation was confirmed in candidate RB-high PDCLs by western blotting for pRB post-PD-0332991 treatment (TKCC-03/-05; figure 1B), with no effect in RB-negative PD-0332991-resistant TKCC-27 cells (figure 1B, right). Interestingly, in RB-high, p16-positive, CDK6-amplified TKCC-26, pRB was reduced post-PD-0332991, suggesting that in PDA CDK6 amplification may support sensitivity to CDK4/6i, irrespective of p16 expression.
Chemotherapeutic GEM synergises with PD-0332991 in RB-high and CDK6-amplified pancreatic PDCLs
Combining PD-0332991 with standard of care therapy in PDA, GEM,7 in PDCLs and primary murine PDA cells (figure 1C) from the genetically engineered Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+ (KPC) model21 22 produced robust cell line-specific efficacy, with combination index analysis23 showing a synergistic interaction between GEM and PD-0332991 in RB-high PDCLs (TKCC-03,-05,-15,-17,-26; figure 1D) and antagonism in RB-low/negative PDCLs (TKCC-06,-16,-23 and -27) and KPC cells (figure 1E). The robustness of synergy was confirmed by examining GEM/PD-0332991 treatment with 25 drug combinations tested on five PDCLs, with synergy repeatedly observed across variable drug ratios in RB-high and antagonism in RB-low/negative lines (figure 1D,E inset, see online supplementary figure 2).
PD-0332991-based monotherapy and combination therapy induce critical effects on RB-high tumour cells
To understand the cellular mechanism of anti-tumour activity of PD-0332991 and its synergy with GEM, we examined the effect of selected treatments on cell cycle and apoptosis in candidate RB-high/RB-negative PDCLs (figure 1F) transduced with FUCCI, a fluorescent, two-colour biosensor of cell cycle progression24 (see online supplementary figure 3). Biosensor function and target modulation were confirmed by live cell imaging of PD-0332991-treated FUCCI-PDCLs (see online supplementary figure 4), showing robust G0/G1 arrest specifically in RB-high TKCC-05, not in RB-negative TKCC-27 cells, confirmed by traditional analysis (see online supplementary figure 4E,F).
Next, PD-0332991 treatment of FUCCI-TKCC-05 significantly induced apoptosis (4–10 days; p<0.0001 and p<0.01, respectively; figure 1G, see online supplementary figure 5). In agreement with published work,25 GEM stimulated apoptosis. Importantly, GEM/PD-0332991 combination significantly increased the apoptotic effect of each monotherapy (figure 1G, see online supplementary figure 5C,E,G), not observed in RB-negative TKCC-27 (figure 1H, see online supplementary figure 5D,F,H), supporting the potentially cytotoxic mechanism of action of PD-0332991 in selected cell types. For comparison purposes, combination of PD-0332991 and clinically used GEM/nab-paclitaxel8 was found to significantly enhance apoptosis in RB-high TKCC-05 (compared with GEM/nab-paclitaxel; see online supplementary figure 5C), although GEM/PD-0332991 effects appeared more robust (across early/late time points, figure 1G).
To examine quiescence-associated alterations in cell cycle dynamics, we used an established flow cytometric methodology26 to analyse FUCCI and quantify G0 versus G1 accumulation post-PD-0332991-based treatments (figure 1I). Both PD-0332991 and GEM/PD-0332991 significantly increased the mKO2++ G0 quiescent fraction,26 compared with GEM or vehicle specifically in the RB-high setting (figure 1J,K; see online supplementary figure 6). Cell cycle analysis of drug-treated apoptotic cells revealed most significant increases in the G0 fraction post-PD-0332991-based therapy (see online supplementary figure 6E). Collectively, these findings provide evidence that activated apoptosis and G0 quiescence are key mechanisms behind the observed synergy between GEM and CDK4/6i in 2D.
CDK4/6 inhibition modulates the invasiveness of RB-high PDA cells and ECM organisation
A single study reported that following incubation with a high PD-0332991 concentration (5 µM), selected commercial PDA lines displayed increased migratory capacity.27 Because both 3D topology and mechanical cues provided by the stroma can shape and drive invasive tumour behaviour,28 we examined the effect of CDK4/6 inhibition on the invasive potential of RB-high and RB-negative PDA cells using an established 3D organotypic model of invasion6 29 (figure 2A). In 3D, PD-0332991 or GEM individually significantly impaired invasion of RB-high TKCC-05 cells, with PD-032291 treatment being more effective (figure 2B, left, middle). Moreover, GEM/PD-0332991 combination was markedly superior to each monotherapy (figure 2B, C). PD-0332991 did not affect the invasive capacity of RB-negative KPC cells in 3D (Figure 2D, E). Further, the observed inhibitory effects on RB-high TKCC-05 invasion were accompanied by decreased proliferation and increased apoptosis in PD-0332991 and GEM/PD-0332991-treated matrices (figure 2F-G, see online supplementary figure 7A,B). In contrast, PD-0332991 treatment slightly increased proliferation of invaded KPC cells, not affecting apoptosis (see online supplementary figure 7C,D). In 2D, GEM/PD-0332991 reduced migratory capacity of RB-high TKCC-05 cells (see online supplementary figure 8), although PD-0332991-mediated effects were more pronounced in 3D, highlighting potential intricacies of CDK4/6 targeting of tumour cells within a complex microenvironment.
Since extracellular matrix (ECM) deposition, fibrosis and signalling processes play a major role in regulating phenotype of pancreatic tumour cells and therapeutic response,6 30 we investigated the effect of CDK4/6i on ECM integrity using primary fibroblast-driven contraction assays,31 as recently achieved by our team (figure 2H).32 PD-0332991 significantly impaired the ability of fibroblasts to contract (figure 2I) and remodel fibrillar collagen, as shown by second harmonic generation (SHG) imaging (figure 2J-K; see online supplementary video 1), and bright-field/polarised light microscopy of Picro-Sirius Red staining (Figure 2L-O), indicating a significantly disorganised ECM network in PD-0332991-treated matrices. Impairment of matrix contraction was independent of changes in fibroblast proliferation (in 2D and 3D; see online supplementary figure 9). As these experiments revealed a significant anti-invasive, antiproliferative and ECM-modulatory activity of PD-0332991 in complex 3D settings, we therefore sought to assess whether PD-0332991 and combinations affect PDA progression and drug response in vivo.
CDK4/6i monotherapy and combination therapy significantly improve survival of mice engrafted with RB-high patient-derived pancreatic tumours
Therapeutic responsiveness in our early-passage PDXs was determined by measuring tumour growth, with experimental design (Figure 3A, see online supplementary figure 10A) enabling assessment of initial therapeutic responsiveness, and longitudinal analysis, comparing intrinsically resistant tumours with those that acquire resistance over time, as previously described.5 33 34 Strikingly, subtype-specific in vivo efficacy was observed, with all three RB-high PDXs examined showing significant long-term inhibition of tumour growth and improvement in overall survival following PD-0332991 monotherapy or GEM/PD-0332991 treatment, compared with standard of care (GEM and GEM/nab-Paclitaxel; Figure 3B-D, see online supplementary table 4A–C). Since GEM/nab-Paclitaxel was associated with cumulative intestinal toxicity when administered over 4–5 treatment cycles, efficacy of targeted therapies was compared with GEM only in the GEM-sensitive TKCC-PDX-26. In addition to improving survival, we observed significantly improved relapse-free survival (defined in online supplementary figure 10) following GEM/PD-0332991, compared with GEM (see online supplementary figure 10). In contrast, RB-negative TKCC-PDX-27, RB-low TKCC-PDX-16 and RB-negative KPC orthografts were resistant to PD-0332991-based therapy (Figure 3E–G, see online supplementary table 5A–C). ‘On-target’ modulation was confirmed by measuring inhibition of pRB and proliferation (Ki-67; see online supplementary figure 11A,B) in PD-0332991-treated RB-high PDX-05.
Analysis of PD-0332991 and GEM/PD-0332991-treated RB-high tumour remnants revealed significant, prolonged in vivo inhibition of proliferation and increased apoptosis (Figure 3H-K), also associated with decreased α-smooth muscle actin (SMA) expression (Figure 3L-M; see online supplementary figure 11C), a prototypical marker of activated pancreatic stellate cells (PSCs), which drive the extensive fibrosis typically observed in PDA.35 36 In PD-0332991-resistant tumours, only GEM/nab-Paclitaxel treatment showed any significant modulatory effects (see online supplementary figure 11D–F). In agreement with immunohistochemistry (IHC) analyses, the significant tumour regression observed in RB-high and CDK6-amplified PDXs post-treatment (30-day time point; see online supplementary figure 11G) further supports the idea that in addition to its cytostatic activity, PD-0332991 may exert cytotoxic effects on specific PDA subtypes in vivo.
PD-0332991-based therapeutic interventions inhibit spread in the liver while sensitising RB-high tumour cells to shear stress and reducing cell attachment
Next, we performed intrasplenic injections of RB-high TKCC-05-FUCCI cells in parallel with systemic PD-0332991 administration as a way of examining efficacy of PD-0332991-based therapies in limiting pancreatic cancer spread to distant sites (Figure 4A). Pathology analyses on serial sections of liver metastases (Figure 4B,C) confirmed that mice treated with PD-0332991 and GEM/PD-0332991 showed a marked reduction in metastatic spread compared with GEM/nab-Paclitaxel. FUCCI analyses of the same liver tissues further confirmed in vivo PD-0332991 activity and ability to effectively inhibit CDK4/6 in liver metastases (Figure 4D, E).
To further understand the observed PD-0332991-mediated reduction of liver colonisation, we examined viability (apoptosis) and cell attachment of RB-high and for comparison, RB-negative PDA cells after exposure to controlled shear stress (100 µL/s, maximum force of 1950 dyn/cm2, Figure 4F),6 37 which CTCs are exposed to in vivo. PD-0332991 significantly induced post-stress cellular apoptosis and decreased cell attachment onto a collagen matrix, specifically in RB-high tumour cells (Figure 4G-I, J-K). Collectively, these experiments suggest a direct role of CDK4/6 targeting in sensitising CTCs already exposed to shear stress as a potential mechanism in which PD-0332991 may modulate metastasis in PDA.
PD-0332991-based therapies significantly improve survival and delay metastasis in metastatic RB-high PDA
To examine the effect of CDK4/6i on PDA tumour progression and metastasis, mice were orthotopically injected with luciferase-TKCC-05 tumours, imaged 1 week post-injection (see online supplementary figure 12A), randomised and treated with standard chemotherapies, PD-0332991 and PD-0332991-based combinations, until ethical endpoint (Figure 5, top).
Critically, PD-0332991-based regimens were significantly more effective than standard therapies (Figure 5A, bottom, see online supplementary table 6) in RB-high metastatic PDA, with GEM/PD-0332991 proving the best therapeutic approach (median survival=117 days, p=0.0001; vs GEM/nab-Paclitaxel median survival=69 days). In addition, metastasis-free survival, monitored by in vivo imaging system (IVIS; see online supplementary figure 12B–C), was significantly increased by PD-0332991-based treatments (see online supplementary figure 13A, supplementary table 7). Given in vitro efficacy was observed when PD-0332991 was combined with GEM/nab-Paclitaxel (see online supplementary figure 5), we also examined this combination in vivo. While treatments were generally well tolerated in experimental animals, the two triple combinations examined using GEM/nab-Paclitaxel and PD-0332991 were associated with some toxicity, namely constipation, which was treated with paraffin oil and controlled by adjusting PD-0332991 dosage. Both regimens, involving concomitant PD-0332991 administration (concomitant) or aiming to assess the potential of CDK4/6i as maintenance therapy in between chemotherapy cycles (maintenance), demonstrated a significant therapeutic benefit over GEM/nab-Paclitaxel (Figure 5A, bottom right, see online supplementary tables 6 and 7), but were not superior to GEM/PD-0332991.
Following initial treatment with GEM/nab-Paclitaxel, a short-term response was followed by progression, measured as increased bioluminescence signal (see online supplementary figure 12C). To examine the therapeutic potential of GEM/PD-0332991 in slowing PDA progression in advanced disease settings, separate cohorts of relapsed animals were subsequently switched to GEM/PD-0332991 or as control, clinically used second-line therapy, capecitabine,38 39 with a superior therapeutic benefit measured with GEM/PD-0332991 (Figure 5B, see online supplementary tables 6 and 7). In concordance, analyses of orthotopic FUCCI-TKCC-05 tumours collected 50 days post-treatment revealed marked quiescence at the primary site (pancreas tumour), regardless of proximity to vessels (Figure 5C-E, see online supplementary figure 13B–C), and no liver metastases in animals treated with PD-0332991 or GEM/PD-0332991 (Figure 5C,F). SHG imaging and polarised light microscopy of Picro-Sirius Red-stained TKCC-05-FUCCI tumours confirmed that PD-0332991-based therapy reduces fibrillar collagen organisation (Figure 5G-H; see online supplementary figure 14A), leads to increased apoptosis and decreased expression of α-SMA in vivo (see online supplementary figure 14B,C).
These data indicate that CDK4/6 inhibition significantly delays pancreatic tumour progression by directly targeting tumour cells (G0 quiescence, apoptosis) and critical components of the microenvironment that have been shown to regulate chemoresistance (ie, decreased stromal activation, reduced fibrillar collagen organisation). As robust efficacy was observed in multiple, independent models of RB-high PDA, RB-based stratification could potentially facilitate a more tailored treatment approach for CDK4-targeting therapy in pancreatic cancer.
Nuclear RB is prevalent in primary operable and metastatic human PDA and has prognostic value
Next, we immunohistochemically examined the expression of RB in human PDA and whether its disruption has prognostic significance (Figure 6A). High RB expression was detected in 64.5% of primary tumour specimens from the APGI/ICGC cohort1 (n=200) and correlated with significantly improved survival (Figure 6B-C, see online supplementary table 8) and lower tumour grade (see online supplementary table 9). Multivariate analysis using a Cox proportional hazards model demonstrated RB loss as an independent poor prognostic factor for disease-specific survival (DSS) (HR 1.552, p=0.0148, table 1). In contrast, p16 expression, examined for comparison purposes, was not prognostic (see online supplementary figure 15A–D). Deregulation of RB signalling has been linked with improved chemoresponsiveness in breast cancer40; however when correlated, RB expression did not specifically associate with survival following adjuvant GEM (see online supplementary figure 15E,F). As expected, RB and p16 levels strongly correlated in the PDX (n=45) and patient tumours from which they were derived (p<0.001, see online supplementary table 10).
The potential prognostic value of RB was subsequently validated in an independent patient NSWPCN cohort20 (n=314), where similar relationships between RB expression and survival were apparent in univariate (Figure 6D-E, see online supplementary table 8) and multivariate analyses (HR 1.364, p=0.02, table 1). As most patients present with advanced stage PDA, we also assessed RB status in metastatic disease (n=54; see online supplementary tables 11 and 12). RB protein was highly expressed in 67% of metastatic specimens. Concordance in RB expression between matched primary and metastatic tumours of PDA, available for 12 of the 54 metastatic samples, was observed in 11 cases (91.7%; Cohen’s Kappa score 0.75; Figure 6F-G) indicating the strength of the agreement as ‘good’.
Collectively, these data demonstrate that a significant proportion of primary operable and metastatic PDA express high levels of the prognostically relevant RB. This druggable, constitutively active pathway may represent an attractive therapeutic target, with immunohistochemical analysis of RB as a potentially simple prognostic and predictive biomarker to guide this therapeutic approach.
Despite significant recent efforts into developing novel treatment combinations,41 chemotherapy remains the standard of care for most patients with advanced pancreatic cancer, despite only modestly improving survival.8 With rapid implementation in the clinical management of specific breast cancers, CDK4/6i are increasingly being tested in other solid tumours.42 43 Here, we examined whether CDK4 pathway inhibition in combination with chemotherapy represented a viable therapeutic approach for pancreatic tumours characterised by G1/S checkpoint aberrations, frequent in the molecular landscape of PDA.1 We demonstrate potent efficacy of single agent and combination CDK4/6i therapy in subtypes of PDA characterised by high RB expression, significantly improving long-term survival while delaying the development of metastasis in robust in vivo models of PDA. We also reveal that the potent efficacy of CDK4/6i monotherapy and combination therapy occurs via a complex mechanism involving deregulation of tumour cell signalling and disruption of the surrounding ECM that collectively drive cancer cell proliferation, invasion and metastasis6 30 44 while revealing a new role of CDK4/6 inhibition in governing tolerance to shear stress, an often overlooked area of premetastatic niche formation for CTCs.45 46
A major area of active investigation is the identification of biomarkers of therapeutic response to CDK4 inhibition. In preclinical models of melanoma and glioblastoma, homozygous loss, methylation or mutation of CDKN2A gene were shown to associate with sensitivity to CDK4/6i,47 48 although in the clinic, CDK4/6i sensitivity may not be strictly defined by p16 status.49 Here, we show that RB expression, but not G1/S checkpoint mutations or p16 levels, may present a robust stratifying biomarker for PD-0332991-based therapy in advanced preclinical models of PDA.
PD-0332991 has traditionally been thought to exert its anti-tumour effects by promoting cytostasis, also supported by limited data in PDA, whereby using 7-day assessment of proliferation as a read-out of PD-0332991 activity, early efficacy has been measured in almost all PDA explants examined.18 Deeper exploration of the long-term responsiveness to PD-0332991 and combination therapy, directly compared with standard of care treatments, in this study highlights the need for a personalised approach in the treatment of pancreatic cancer, where RB-high PDA tumours respond long term to PD-0332991-based therapy in vivo and RB-low/negative tumours do not. Moreover, building on recent studies in other cancers, which suggest a more complex mechanism of action for CDK4/6i, including inhibition of EMT signalling in breast cancer metastasis50 and improved anti-tumour immunity,51 our study provides additional new evidence that PD-0332991 displays multifaceted effects on tumour cells (quiescence, apoptosis), alters ECM organisation and reduces tolerance to shear stress (Figure 4), thereby having a global effect in this aggressive disease, not examined previously.
Using a combination of 2D, 3D in vitro and in vivo models, we specifically show that PD-0332991 significantly induces apoptosis in RB-high pancreatic tumour settings and also enhances the apoptotic effect of chemotherapeutic GEM, a well-tolerated and clinically used agent in PDA (figures 1-3). Monitoring the onset of liver metastasis in vivo revealed that PD-0332991 monotherapy and combination therapy significantly impaired liver colonisation in the RB-high setting (Figure 4A-E), delaying disease progression in a metastatic PDA model (Figure 5). Of note, CDK4/6i may potentially be a beneficial second-line therapy, following progression on GEM/nab-Paclitaxel in RB-high PDA, as our in vivo findings suggest (Figure 5B). Given the high rate of chemotherapeutic resistance in advanced patients with PDA treated with these chemotherapies and their subsequent extremely poor outcome, pending further preclinical study, CDK4/6i-based approaches may potentially be a viable second-line treatment for specific subtypes of advanced PDA.
CDK4/6 inhibition modulated ECM organisation in vitro and in vivo (Figures 2,5), with in vivo responses to PD-0332991-based therapy associated with decreased expression of α-SMA, a marker of activated PSCs and stromal activation. As our in vitro findings suggest that the effects on ECM are independent of the antiproliferative activity of PD-0332991, it is plausible to hypothesise that changes in ECM mechanics induced by PD-0332991 may directly disrupt PSC activation, the subsequent tumour cell–PSC crosstalk and ultimately further improve therapeutic sensitivity. Indeed, recent work has shown that matrix stiffness and organisation can directly regulate differentiation and migration of PSCs.52
PD-0332991 and GEM strongly synergised irrespective of p16 or p53 status (eg, whether pancreatic tumour harboured an inactivating TP53 mutation (C124*; PDX-26), mutation contributing to partial rescue (V173M; PDX-03) or higher than wild-type p53 activity (G245S; PDX-05).53 CDK4/CDK6 amplification or overexpression has been associated with in vitro CDK4/6i resistance,47 although CDK4-amplified liposarcomas are sensitive to PD-0332991.54 Interestingly, the CDK6-amplified PDX-26 and matched PDCL responded to PD-0332991-based treatment, suggesting that in PDA CDK6 amplification in the context of functional RB does not preclude effective CDK4 pathway inhibition. Although CDK6 is strongly functionally linked to CDK4, this enzyme may exert other kinase-independent functions in regulation of angiogenesis and/or cell differentiation,55 representing a potentially separate thus far unexamined role for CDK6 inhibition in PDA therapy.
Several smaller studies have investigated the prognostic role of RB in PDA, with inconsistent findings.56 Here, RB protein was highly expressed in a significant proportion of PDA (65%, APGI/ICGC; 43%, NSWPCN; 67%, metastatic cohort), suggesting that this could be a viable therapeutic target for tailored CDK4/6i-based treatments that act on this signalling network. Moreover, the observed concordance in RB levels in the small set of available matched primary and metastatic tissue lends hope that metastases may still be co-targeted with this type of therapy. Loss of RB protein was associated with poor patient outcome, with RB an independent prognostic factor for DSS in two sizeable independent PDA cohorts (table 1). RB immunohistochemistry was a clean nuclear stain without considerable background. In our hands, it appeared comparable with estrogen receptor (ER) staining in terms of interpretation of intensity within processed material. Pending further validation, the use of a tissue-based assay for RB expression could be adapted for clinical application in diagnostics to, combined with other markers, help identify those with poorest prognoses and also potentially identify a cohort of patients to trial CDK4/6i.
In conclusion, we provide preclinical proof-of-concept for the efficacy of a personalised treatment strategy using CDK4/6i to treat RB-high PDA. We reveal key mechanisms whereby CDK4-targeting therapy may regulate multiple cellular processes and environmental cues within the primary tumour, secondary site and during CTC transit, which collectively govern pancreatic cancer progression. We hope that with two non-biomarker-driven phase I/II trials of PD-0332991 in pancreatic cancer underway (NCT02501902, NCT03065062), these findings may have complementary and potentially significant therapeutic implications for the personalised treatment of PDA in future.
The authors would like to thank consumer representatives Professor Ros Pesman, Mr Benjamin Bravery and Ms Jan Mumford for their involvement in this project and a critical review of the manuscript. Dr Chou would also like to acknowledge Associate Professor Adrienne Morey for mentorship during PhD studies.
Contributors Study concept and design (MP, AJG, AC, SJC, PT); data acquisition (AC, DF, AMN, VTC, DW, AP, AS, RS, AD, KJM, JT); analysis and data interpretation (AC, DF, DW, AP, TRC, TJM, JT, CV, KJM, AB, OJS, JM); drafting of manuscript (AC, DF, MP); critical review and edit of manuscript (AJG, PT, AJ, SJC, JS, NW, SMG, LAC, AB, TRC, AVB, TJM, OJS, JM); patient cohort data (APGI, AJ, AVB, JS, AJG, JT, SMG); statistics (AC, MC, MP, DKC); obtained funding (MP, AJG, AC, SJC, PT, JS, AMN, AB, NW); study supervision (MP, AJG).
Funding This work was supported by the National Health and Medical Research Council of Australia (NHMRC) Project Grants 1081312 and 1105640, Cancer Australia Project Grants 1060522 and 1100722 with fellowship support from Cancer Institute NSW, Australian Research Council and NHMRC, scholarship support from NHMRC and Sydney Catalyst and philanthropic support from the Avner Pancreatic Cancer Foundation, The Philip Hemstritch Fellowship in Pancreatic Cancer; Len Ainsworth Pancreatic Cancer Fellowship, Estate of the late RT Hall Cancer Gene Discovery and Validation Program and Norman Green Support Grant.
Competing interests None declared.
Patient consent Detail has been removed from this case description/these case descriptions to ensure anonymity. The editors and reviewers have seen the detailed information available and are satisfied that the information backs up the case the authors are making.
Ethics approval Ethics approval for acquisition of data and biological material was obtained from human research ethics committees from each participating institution (Sydney Local Health District Human Research Ethics Committee X11-0220). In vivo experiments were approved by Garvan/St Vincent’s animal ethics committee (14/11, 14/12).
Provenance and peer review Not commissioned; externally peer reviewed.