Preclinical Drug Testing of the CDK 4/6 Inhibitor Palbociclib in Combination With a PI3K or MEK Inhibitor in Colorectal Cancer Cell Lines

Background: Studies have demonstrated the efficacy of Palbociclib (CDK 4/6 inhibitor), Gedatolisib (PI3K/mTOR dual inhibitor) and PD0325901 (MEK1/2 inhibitor) in colorectal cancer (CRC), however single agent therapeutics are often limited by resistance. The main purpose of this in vitro study is to comprehensively test two drug combinations [Palbociclib with Gedatolisib (P+G) and Palbociclib with PD0325901 (P+PD)] to determine the most synergistic combination for clinical development. Methods: We compared the anti-proliferative effects of both drug combinations in five CRC cell lines with various mutations (Caco-2, DLD-1, LS1034, SNUC4 and LS411N). Reverse Phase Protein Arrays was used to investigate the effects of P+G on the total and phosphoproteins of the signalling pathways. Results: Our results from toxicology experiments indicated that the P+G is a superior combination. The combination of P+G had synergistic anti-proliferative effects in all cell lines [CI range: 0.11-0.69]. The combination of P+PD is also synergistic in all cell lines [CI range: 0.06-0.44], except LS411N with BRAF V600E mutation [CI=14.7]. The combination of P+G caused significant suppression of S6rp(S240/244) in all cell lines, without AKT reactivation. This indicated efficient blockage of PI3K/AKT/mTOR pathway, even in PIK3CA mutated cell lines. The combination of P+G induced BAX and Bcl-2 levels in PIK3CA mutated cell lines. The combination of P+G caused MAPK/ERK reactivation, as seen in total EGFR increase and this was not mutationally exclusive. Conclusion : This in vitro study demonstrated that the combination of P+G has synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines. This data provides good rationale for further in vivo studies for P+G novel combinative therapy in CRC. Separately, the S6rp(S240/244) may serve a promising biomarker of responsiveness.


BACKGROUND
Colorectal cancer (CRC) is the second most common cause of cancer death in Europe. It is estimated that the global burden of CRC will increase by 60%, with 2.2 million new cases and one million deaths worldwide by 2030. 1 In the metastatic CRC setting, compound agent chemotherapies given in combination with the anti-EGFR (epidermal growth factor receptor) or anti-VEGF (vascular endothelial growth factor) monoclonal antibodies are standard clinical practice, providing improvement in patients outcomes reaching median OS between 29 and 36 months (in wild-type). 2,3 However, more than 50% of cases would eventually relapse and subsequent treatment options rarely offer high clinical impact, especially in patients with RAS mutations. 4 Treatment for CRC is complexed and often limited by resistance, which can be intrinsic or acquired. The crosstalk between the PI3K/AKT/mTOR (phosphatidylinositol-3-kinase/ acutely transforming retrovirus/mammalian target of rapamycin) and MAPK/ERK (mitogen activated protein kinase) pathways has been recognised as a key mechanism of resistance in Oncology therapy. 5 Other mechanisms that lead to resistance evolution include: 1) Formation of new secondary site resistance mutations within the target kinase; 2) Activation of escape bypass routes involving pathways such as the MAPK/ERK and PI3K/AKT/mTOR; 3) Dysregulation of downstream effectors; 4) Transformation into pro-metastatic phenotypes which enable the cancer cells to survive the effects of treatment; 5) Immune adaption within the tumour microenvironment to enable cancer cell survival, via either immune-dependent or immune-independent processes. 6 Theoretically, when compared to the single agents, combined therapies can produce synergistic inhibition in a relatively safe manner to reduce multiple growth signal transmission responsible for development of drug resistance. There is now a growing appreciation for using the combinative therapeutic approaches which can be exploited through multiple modalities such as radiotherapy, chemotherapy, immunotherapy or targeted agents.
The PI3K/AKT/mTOR and MAPK/ERK signalling pathways are highly implicated in CRC pathogenesis with key mutations like RAS, BRAF and PIK3CA arising from both pathways. There are data from Phase I/ II trials to support the use of Palbociclib (CDK 4/6 inhibitor) 7-9,10,11 , Gedatolisib (PI3K/mTOR dual inhibitors) 7,12,13 and PD0325901 (selective MEK1/2 inhibitor) [14][15][16] in various types of cancers. These inhibitors failed to cause significant tumour regression when used as single agents because of resistance. As shown in breast cancer models, the combination of CDK 4/6 inhibitor with a PI3K/mTOR inhibitor can produce synergistic effects. This specific drug combination can overcome treatment-related resistance by preventing RSK activation and subsequent MAPK/ERK pathway activation. [17][18][19][20] Currently, there are three active Phase I trials evaluating the combination of Palbociclib with Gedatolisib in patients with refractory malignancies including CRC. [7][8][9] Similarly, another Phase Ib trial is evaluating the effectiveness of combining a different PI3K/mTOR dual inhibitors (LY3023414) with a CDK4/6 inhibitor (Abemaciclib) in multiple common cancers. 21 The combined approach of Palbociclib with Gedatolisib to prevent the emergence of resistance in breast cancer, is hypothetically applicable to other cancers including CRC. In parallel, there is strong preclinical evidence for evaluation of co-inhibition using Palbociclib with PD0325901 in CRC. [14][15][16] The combination of Gedatolisib with PD0325901 is toxic in humans and not advisable as previously shown. 22 In summary, combined drug inhibition have multiple advantages over single agents since it can maximise anti-proliferative efficacy within an acceptable overlapping drug toxicity limit. In comparison to single agents, the combined drug therapies exhibit multiple-target inhibitions and cellular regulatory actions, thus more likely to be effective in attenuating drug resistance pathways. This method has been exploited in various cancers, particularly in breast cancer. As we have noted there is a research-gap in CRC, therefore we want to explore novel therapeutics using combinative drug approaches. We hypothesise that the combination of Palbociclib with either Gedatolisib or PD0325901 can produce synergistic benefits with potential clinical application in refractory CRC.

Aims
Specific Aim 1: To demonstrate in vitro that Palbociclib can act synergistically with either Gedatolisib or PD0325901 as reflected by improved anti-proliferative effects in five CRC cell lines with different mutational backgrounds. This preclinical information will be utilised for future in vivo evaluation and clinical trial efforts. 7 from Selleckham. The drugs were prepared in 100% dimethylsulfoxide (DMSO) at the stock concentrations of 10mM, 5mM and 10mM, respectively. The two drug combinations tested were Palbociclib with Gedatolisib (P+G) and Palbociclib with PD0325901 (P+PD).

Proliferation Assays and Drug Combination Analysis
The acid phosphatase assay was used to test the anti-proliferative effects of Palbociclib, Gedatolisib and PD0325901, alone and in combination in each cell line. This was performed over 6-day period.
Cell were plated at 1x10 4 cells/mL into 96-well plates (100µL per well) and incubated at 37°C with 5% CO2 for 24 hours. 200µL of sterile H2O was added around the edges of the plate to prevent it from drying out.
Following 24 hours incubation, drugs were added at the indicated concentrations and incubated at 37˚C for 5 days. On Day 6, all drug was removed, washed and processed for absorbance measurement at 405nm using a 96-well plate reader. Inhibition of proliferation was calculated relative to untreated controls to obtain the dose of half maximal inhibitory concentration (IC50). The individual inhibitors IC50 values were used for dosing guidance in the drug combinations analysis. The doses used for the two drugs combinations in each cell lines are listed in Table 1.

Protein extraction from cell lines
For RPPA investigations, we selected the Palbociclib and Gedatolisib because both drugs demonstrated synergism as combinations in all the cell lines tested in our study. The Caco-2, DLD-1, LS1034 and SNUC4 cell lines were selected. The cell lysates were prepared according to the MD Anderson protocol as previously described by us 23,24 . The complete methods of protein extraction from cell lines are detailed in Additional File 2.

Reverse Phase Protein Array (RPPA)
Cell lysates with protein concentration of 1.5µg/µL for each replicate were prepared. Before RPPA processing, each sample was solubilised

Effects of Palbociclib, Gedatolisib and PD0325901 in CRC cell lines
The corresponding IC50 values of single-agent Palbociclib, Gedatolisib and PD0325901 in CRC cell lines are summarised in Table 1

PD0325901 (P+PD) in CRC cells lines
Drug combination analysis showed that combination P+G has a synergistic anti-proliferative effect in all CRC cell lines tested [refer Table 2; Figure 1]. The combination of P+G is very synergistic in [refer Table 2; Figure 2].

RPPA Analysis
We conducted RPPA analysis using 40 primary antibodies representing multiple nodes of the PI3K/AKT/mTOR, MAPK/ERK and intracellular apoptotic signalling pathways, following 30 minutes and 4 hours treatment with Palbociclib, Gedatolisib and the combination in the Caco-2, DLD-1, LS1034 and SNUC4 cell lines. We selected Palbociclib and Gedatolisib because this drug combination was synergistic in all cell lines tested in our cell proliferative study.
Overall, there was little difference between the protein levels measured at the 30-minute and 4-hour timepoints following exposure to 4-hour timepoint.
We also aimed to investigate the proteomic effects of the combination of Palbociclib with Gedatolisib (P+G) in the CRC cell lines with various mutational backgrounds to identify potential biomarker(s) for this novel therapy.
Emerging experimental data has suggested utilising PI3K/mTOR inhibitors to induce non-cell autonomous actions by modulating signal transduction during G1 to S phase, leading to increased cell death. [42][43][44][45] Interestingly, we did not observe any increase in pRb(S807/S811) following treatment with P+G in combination. This is consistent with what was previously described by Vora et al. 46 It appeared that PI3K inhibition suppress AKT phosphorylation, but sometime failed to suppress CDK 4/6 activity, as measured by Rb phosphorylation 47 . Nonetheless, the regulation of Rb function by phosphorylation during cell cycle is not fully understood. Rb in mammalian cells has sites is required for the ability of Rb to regulate apoptosis. 48,49 As shown in in vitro, Rb may be phosphorylated at few additional sites (S608, S795) in addition to S807/S811 in the role of apoptosis. 50 Furthermore, there is evidence to suggest that dephosphorylation of Rb has been widely observed during apoptosis. 51  Finally, we observed EGFR and RSK upregulation in all cell lines after 4 hours of drugs treatment, which may be associated with upstream MAPK/ERK reactivation.
Total EGFR and RSK upregulation were observed with both single agents and combination therapy in some of the cell lines. This suggests that the mechanism promoting resistance to PI3K-targeted inhibitors (which was Gedatolisib in this study) include feedback loops which lead to reactivation of membrane RTKs and the contralateral MAPK/ERK pathway. This further supports our hypothesis that multiple target inhibition strategy, rather than single agent, is better for tackling resistance occurrence.
It is important to note the limitations of our study. Firstly, it was an in vitro study and limited to five cell lines. Secondly, not all specific exon mutations were tested in this study, specifically PIK3CA mutations in exon 20 which may be biologically more relevant than exon 9 mutations from an epidemiology standpoint. [52][53][54] Thirdly, the RPPA analysis with 40 preselected antibodies was performed at only two timepoints (i.e. 30 minutes and 4 hours) post drug exposure. The specific mechanism of synergism for the combination of P+G could not be completely defined; however, several possible mechanisms were observed by our RPPA analysis, including more complete inhibition of protein synthesis-related signalling e.g. S6rp(S240/S244) and increased activation of early apoptotic signalling. Despite these limitations, our study has produced evidence to support further in vivo evaluation, which is in progress.

CONCLUSION
In summary, the novel combination of Palbociclib with Gedatolisib (P+G) displays clear synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines, relative to the single agents. Our results offer good rationale for further in vivo study and clinical development of P+G as emerging therapeutics in metastatic CRC patients. S6rp(S240/S244) may be a marker of responsiveness for this novel combination therapy.

CONSENT FOR PUBLICATION
Not applicable

AVAILABILITY OF DATA AND MATERIAL
All data generated or analysed during this study are included in this published article and its supplementary files.

COMPETING INTERESTS
The authors declare that there is no competing interest.