Abstract 2815: Antitumor activity of CDK4/6 inhibition in combination with radiation therapy on glioblastoma stem cells

Purpose: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors like palbociclib, ribociclib, and abemaciclib have improved progression free survival in patients with metastatic, estrogen receptor positive (ER+) breast cancer, acquired resistance to these drugs limits their efficacy. Despite promising new studies defining the utility of CDK4/6 inhibitors in the upfront, non-metastatic setting, there is limited data available on the effects of concurrent CDK4/6 inhibition and radiation (RT). Methods: Transcriptomic and proteomic expression data was used to quantify changes in RNA and protein expression in ER+ breast cancer cell lines (MCF-7, T47D) after short term (16 hour) CDK4/6 inhibition. Proliferation assays were used to determine the half-maximal inhibitory concentration (IC50) of palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays were performed to calculate the radiation enhancement ratio (rER) and the surviving fraction at 2 Gy for each treatment. Homologous recombination (HR) proficiency was assessed using RAD51 and γH2AX foci formation and a pYFP reporter was used to assess non-homologous end joining (NHEJ) efficiency. Western blots were used to quantify protein expression. MCF-7 xenografts were used to study the efficacy of combination (palbociclib + RT) therapy in vivo. MCF-7 and T47D cell lines with acquired resistance to CDK4/6 inhibition (IC50 >1uM) were used for comparison in all assays. Results: Transcriptomic and proteomic analyses identified changes in expression of DNA damage response mediators and cell cycle machinery with short term CDK4/6 inhibition. Palbociclib significantly radiosensitized ER+ cell lines at concentrations at or below the IC50 value in clonogenic survival assays (MCF-7 rER: 1.22-1.52, T47D rER: 1.23-1.50) and led to a decrease in the surviving fraction of cells at 2 Gy (p < 0.001). Similar results were observed in ribociclib- (rER: 1.08 - 1.68) and abemaciclib-treated (rER: 1.19 - 2.05) cells. MCF-7 and T47D cells treated with CDK4/6 inhibition and RT showed a decrease in RAD51 foci formation, suggesting a decrease in HR efficiency (p < 0.001). However, CDK4/6 inhibition did not affect NHEJ efficiency (p > 0.05). CDK4/6 inhibition + RT led to a decrease in expression of protein expression of HR meditators like p-CHK1 but did not affect phosphorylation of NHEJ proteins like pKu80/pKu70. Cells with acquired resistance to CDK4/6 inhibition did not demonstrate radiosensitization (MCF-7 rER: 0.93 - 1.03, T47D rER: 0.96 - 1.11) or changes in RAD51 foci formation with combination treatment. Conclusions: Our data suggests that CDK4/6 inhibitor-mediated radiosensitization may be effective in ER+ breast cancers prior to the development of CDK4/6 inhibitor resistance. These studies provide preclinical rationale to test CDK4/6 inhibition + RT in women with locally-advanced ER+ breast cancer at high risk for locoregional recurrence. Citation Format: Andrea M. Pesch, Nicole Hirsh, Benjamin C. Chandler, Anna R. Michmerhuizen, Cassandra L. Ritter, Marlie Androsiglio, Kari Wilder-Romans, Meilan Liu, Christina L. Gersch, Jose M. Larios, James M. Rae, Corey W. Speers. CDK4/6 inhibitor-mediatated radiosensitization of estrogen receptor positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6280.

Aberrant cell cycle progression is a hallmark feature of cancer cells. Cyclin-dependent kinase 4 and 6 (CDK4/6) drive progression through the G1 stage of the cell cycle, at least in part, by inactivating the tumor suppressor, retinoblastoma (RB). CDK4/6 are targetable and the selective CDK4/6 inhibitor, palbociclib (IBRANCE/PD-0332991), was recently FDA approved for the treatment of estrogen receptor-positive, HER2-negative advanced breast cancer. In cutaneous melanoma, driver mutations in NRAS and BRAF signal to promote CDK4/6 activation suggesting that inhibitors such as palbociclib are likely to provide therapeutic benefit most likely in combination with BRAF and/or MEK inhibitors that are FDA-approved. However, the determinants of the response to CDK4/6 inhibitors alone and in combination with other targeted inhibitors are poorly defined. Furthermore, in vivo systems to quantitatively and temporally measure the efficacy of CDK4/6 inhibitors and determine the extent that CDK activity is reactivated during acquired resistance are lacking. Here, we describe the heterogeneous effects of CDK4/6 inhibitors, the expression of anti-apoptotic proteins that associate with response to CDK4/6 and MEK inhibitors, and the development of a luciferase-based reporter system to determine the effects of CDK4/6 inhibitors alone and in combination with MEK inhibitors in melanoma xenografts. These findings are likely to inform on-going and future clinical trials utilizing CDK4/6 inhibitors in cutaneous melanoma. Citation Format: Jessica L. Teh, Timothy Purwin, Evan J. Greenawalt, Inna Chervoneva, Allison Goldberg, Michael E. Davies, Andrew Aplin. An in vivo reporter to quantitatively and temporally analyze the effects of CDK4/6 inhibitor-based therapies in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5199.

The phosphatidylinositol-3-kinase (PI3K) pathway is mutated and aberrantly activated in breast and other cancers and plays a key role in cancer cell proliferation and survival.1 2 The PI3K pathway is deregulated through a variety of mechanisms, including mutation or amplification of PI3K, loss or inactivation of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 ( PTEN ), as well as activation of tyrosine kinase growth factor receptors or oncogenes upstream of PI3K.3 4 Activating mutations in PIK3CA , the gene encoding the alpha isoform (p110 α) catalytic subunit of PI3K, is present in up to 40% of patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers5 6 and represents a molecular target to personalise therapy of selected patients with breast cancer.2 Standard of care therapy for advanced HR-positive/HER2-negative breast cancers consists on endocrine therapy with or without the use of cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors.7 Therapy-resistance inevitably occurs in the majority of patients. The rationale of combining PI3K inhibitors and endocrine therapy is to synergistically inhibit both the PI3K and ER pathways.8 Initial trials of pan-PI3K inhibitors plus endocrine therapy for patients with advanced breast cancer showed modest benefit with high rates of toxicity limiting their clinical drug development.9–11 Selective isoform-specific PI3K inhibitors, such as an α-specific PI3K inhibitor, have subsequently revealed activity with less toxicity.12 13 The phase III SOLAR-1 (Clinical Studies of Alpelisib in Breast Cancer 1) trial investigated the efficacy and safety of alpelisib, a α-specific class-I PI3Kinhibitor, plus fulvestrant versus placebo plus fulvestrant in patients with metastatic HR-positive/HER2-negative breast cancer who had received endocrine therapy beforehand.14 About 85.6% of patients …

G1T28-1 is a clinical stage, small molecule inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6). Hematopoietic stem and progenitor cells (HSPC) require CDK4/6 for proliferation, allowing the transient arrest of HSPC in the G1 phase of the cell cycle by CDK4/6 inhibition. The CDK4/6 transient arrest may reduce the sensitivity of HSPC to DNA damaging chemotherapies by limiting G1 to S-phase progression in the setting of unrepaired DNA damage. A reduction in chemotherapy-induced HSPC death would in turn reduce chemotherapy-induced myelosuppression (CIM), the major dose-limiting toxicity of most cytotoxic anti-cancer agents. G1T28-1 is highly potent, exhibits exquisite selectivity, and has favorable pharmacology allowing the induction of a predictable and well-defined transient arrest of HSPC with greater uniformity and distinct profile from the arrest induced by less potent and selective CDK4/6 inhibitors. To characterize HSPC inhibition induced by G1T28-1 treatment, we studied the compound's pharmacodynamic properties in mice. Murine HSPC proliferation was measured in vivo using EdU incorporation. This work showed that G1T28-1 induces a significant, rapid and reversible G1 arrest in all early hematopoietic lineages in a dose-dependent manner. Above a determined threshold dose, higher doses do not augment the percentage of specific HSPC fractions in G1, but instead extend the duration of the block of G1 to S-phase traversal of these populations. The degree of G1 arrest differed for specific HSPC fractions, multipotent progenitors, and early lymphoid progenitors being more sensitive than populations of early myeloid and erythroid progenitors. To determine the ability of G1T28-1 to ameliorate CIM, we assessed the effect of concomitant G1T28-1 administration in a well-characterized murine model of 5FU-induced myelosuppression. A single dose of G1T28-1 given 30-minutes prior to 5FU provided multilineage protection resulting in a more rapid recovery of complete blood counts (CBCs) compared to animals who received 5FU + vehicle. This effect persisted through multiple cycles of 5FU. After four cycles of 5FU, even at count recovery, the CBC's in animals treated with vehicle + 5FU were significantly worse compared to cycle one, whereas this chronic myelosuppressive effect was completely ameliorated in animals that received G1T28-1 prior to each 5FU dose. In conclusion, we have demonstrated that G1T 28-1 is a highly potent CDK4/6 inhibitor that causes robust and transient inhibition in a broad range of HSPC. Arrest at the time of 5FU administration significantly lessens CIM; benefitting all hematopoietic lineages and attenuating the acute myelosuppression of each cycle of chemotherapy as well as reducing a chronic reduction in peripheral blood counts caused by repeated, serial chemotherapy dosing. Citation Format: Jessica A. Sorrentino, Shenghui He, John E. Bisi, Patrick J. Roberts, Jay C. Strum, Norman E. Sharpless. G1T28-1, a novel CDK4/6 inhibitor, protects murine hematopoietic stem and progenitor cells from cytotoxic chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 941. doi:10.1158/1538-7445.AM2015-941

Purpose: Fractionated radiation (RT) is used in the adjuvant setting for locoregional control and to prevent the development of metastatic lesions in estrogen receptor positive (ER+) breast cancers. We previously demonstrated that inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) along with RT leads to the radiosensitization of ER+ breast cancers, but the exact mechanism by which this occurs is unknown. We hypothesized that the presence of RB is necessary for effective double strand repair of radiation-induced DNA damage mediated through homologous recombination (HR), and that this is prevented using CDK4/6 inhibition. Methods: Pharmacological CDK4/6 inhibition was achieved using three FDA approved CKD4/6 inhibitors: palbociclib, ribociclib, and abemaciclib. Genetic knockdown of RB1 was performed using siRNA and knockout was achieved using a CRISPR-Cas9 system. Parental and RB1 knockdown/CRISPR MCF-7 and T47D cells were treated for 72 hours to assess drug sensitivity. Cells were pretreated with a CDK4/6 inhibitor one hour prior to RT and colony formation was quantified to assess changes in radiosensitivity. MCF-7 cells expressing an HR-specific GFP reporter were used to assess HR competency. RB, yH2AX, and RAD51 cellular localization following RT and CDK4/6 inhibition was assessed using immunofluorescence assays. Flow cytometry with propidium iodide staining was used to assess cell cycle distribution. Protein expression was assessed by immunoblotting. Results: CDK4/6 inhibition with palbociclib, ribociclib, and abemaciclib + RT radiosensitizes ER+ breast cancer cells at sub-IC50 concentrations in vitro (rER: 1.21 – 2.05) through impaired HR, which we confirmed using MCF-7 cells that express a stable HR-GFP reporter system (p < 0.01). RB1 knockdown decreased single-agent efficacy of CDK4/6 inhibition on the proliferation of ER+ breast cancer cell lines, leading to an increase in the IC50 for each CDK4/6 inhibitor. Palbociclib, ribociclib, and abemaciclib failed to induce G1 cell cycle accumulation after RB1 knockdown (p > 0.05). Genetic knockdown of RB1 led to a decrease in the ability of breast cancer cells to perform HR-directed DNA repair (p < 0.01) independent of drug treatment, and further suppression of HR with CDK4/6 inhibition was lost in MCF-7 and T47D cells lacking RB expression (p > 0.05). Furthermore, RB protein is necessary for CDK4/6i mediated radiosensitization as evidenced by the abrogation of radiosensitization in RB null isogenic models of ER+ breast cancer (MCF-7 rER: 0.97 ± 0.13). Conclusions: Our data suggests that CDK4/6 inhibitor-mediated radiosensitization and HR suppression is dependent on RB expression. Thus, RB might serve as an effective biomarker for patient selection in future clinical trials that seek to combine CDK4/6 inhibition + RT. Citation Format: Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Benjamin C. Chandler, Kari Wilder-Romans, Meilan Liu, Erin Cobain, Lori J. Pierce, James M. Rae, Corey Speers. RB loss mitigates CDK4/6 inhibitor-mediated radiosensitization of estrogen receptor positive (ER+) breast cancers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-028.

Simple SummaryCyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used to treat metastatic hormone receptor-positive/human epidermal growth factor receptor 2-negative breast cancer. Despite the effectiveness of CDK4/6 inhibitors, acquired resistance occurs in almost all cases. Strategies to address this issue have not been developed yet. We investigated mechanisms of resistance to CDK4/6 inhibitor in breast cancer and potential therapeutic strategies. We found that cyclin E-CDK2 mediated phosphorylation of C-MYC is responsible for resistance to CDK4/6 inhibitor by suppressing C-MYC induced senescence. On the contrary, the synergistic anti-proliferative effect of the combined inhibition of CDK2 and CDK4/6 overcomes acquired resistance to CDK4/6 inhibitors by enhancing senescence. Our findings could pave the way for the development CDK2-specific kinase inhibitor for the treatment of breast cancers that are resistant to CDK4/6 inhibitor.Breast cancer represents the number one global cancer burden in women and the hormone receptor (HR)-positive subtype comprises approximately 70% of breast cancers. Unfortunately, acquired resistance ultimately occurs in almost all cases, even though cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are a highly effective therapy for HR-positive/human epidermal growth factor receptor 2-negative subtype. Here, we investigated mechanisms of resistance to CDK4/6 inhibitor and potential therapeutic strategies using our palbociclib-resistant preclinical model. We observed that cyclin E was significantly overexpressed in palbociclib-resistant cells, and similar association was also confirmed in pleural effusion samples collected from HR-positive breast cancer patients. After confirmation of cyclin E-CDK2 interaction by co-immunoprecipitation, we demonstrated CDK2 inhibition combined with palbociclib synergistically suppressed proliferation of palbociclib-resistant cells and growth of palbociclib-resistant xenograft in mice. We also proved that enhancing C-MYC-mediated senescence is a novel mechanism behind the synergism created by targeting both CDK2 and CDK4/6. Furthermore, the clinical relevance of cyclin E as a therapeutic target was supported by significant association between CCNE1 overexpression and poor prognosis based on large-scale public gene expression data sets in HR-positive breast cancer patients. Therefore, we propose cyclin E-CDK2 signaling as a promising therapeutic target for overcoming cyclin E-associated resistance to CDK4/6 inhibitor.

Dysregulation of the cell cycle is a hallmark of nearly all cancers, and efforts to target signaling pathways regulating cell growth and proliferation have driven much of cancer drug discovery. Despite advances in novel therapeutics, however, most patients with advanced neoplasms do not achieve long-term survival with single agent targeted therapy. Here, we describe a novel “triple inhibitor” (i.e., SRX3177) that simultaneously targets three oncogenes promoting cancer cell growth: phosphatidylinositol-3 kinase (PI3K), cyclin-dependent kinases 4 and 6 (CDK4/6), and the epigenetic regulator BRD4. This rationally-designed, thieno-pyranone scaffold-based small molecule inhibitor uses known synthetic lethality relationships to orthogonally disrupt three targets within the cancer cell with one agent. Single agent CDK4/6 inhibitors such as palbociclib, which is FDA-approved in combination with hormone therapy in estrogen-receptor positive breast cancer, suffer from being cytostatic in nature, requiring combinations to be more effective and avoid development of resistance. Concurrent PI3K inhibition can prevent resistance to CDK4/6 inhibition, and combined CDK4/6 and PI3K inhibition leads to synthetic lethality reported in a number of cancer types, including breast cancer and mantle cell lymphoma. Moreover, blocking the chromatin reader protein BRD4 downregulates MYC and cyclin D1 transcription, further promoting cell cycle arrest in G1. Thus, we designed SRX3177 as a triple inhibitor of PI3K, CDK4/6, and BRD4 to maximally block cell cycle progression and cancer cell growth. SRX3177 is a potent ATP competitive CDK4/6 inhibitor (IC50: CDK4 = 2.54 nM, CDK6 = 3.26 nM), PI3K inhibitor (IC50: PI3Kα = 79.3 nM, PI3Kδ = 83.4 nM), and BRD4 inhibitor (IC50: BD1 = 32.9 nM, BD2 = 88.8 nM). In a panel of mantle cell lymphoma, neuroblastoma, and hepatocellular carcinoma cell lines, SRX3177 has maximal IC50 values of 578 nM, 385 nM, and 495 nM respectively. This represents a 19 to 82-fold increase in potency compared to palbociclib. SRX3177 is 5-fold more potent in cancer cells than the combination of similar potency single PI3K, CD4/6, and BRD4 inhibitors (i.e., BKM120 + palbociclib + JQ1). SRX3177 is also 40-fold less toxic to normal epithelial cells than the co-treatment with single inhibitors. Furthermore, SRX3177 induces cell cycle arrest and apoptosis in propidium iodide and annexin V assays, respectively. Finally, SRX3177 inhibits Akt and Rb phosphorylation (downstream of PI3K and CDK4/6 signaling, respectively) and blocks BRD4 binding to chromatin. Thus, our triple inhibitor SRX3177 is efficacious, is more potent and less toxic to normal cells than administration of three individual inhibitors, and has robust pharmacodynamic effects on its targets. Taken together, our data support the development of SRX3177 as a novel therapeutic agent for multiple cancers. Citation Format: Adam M. Burgoyne, Francisco M. Vega, Alok Singh, Shweta Joshi, Joseph R. Garlich, Guillermo A. Morales, Tatiana G. Kutateladze, Donald L. Durden. The novel triple PI3K-CDK4/6-BRD4 inhibitor SRX3177 harnesses synthetic lethality relationships to orthogonally disrupt cancer cell signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-298. doi:10.1158/1538-7445.AM2017-LB-298

BackgroundBreast cancer is the global leading cancer burden in women and the hormone receptor-positive (HR+) subtype is a major part of breast cancer. Though cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are highly effective therapy for HR+ subtype, acquired resistance is inevitable in most cases. Herein, we investigated the paternally expressed gene 10 (PEG10)-associated mechanism of acquired resistance to CDK4/6 inhibitors.MethodsPalbociclib-resistant cells were generated by exposing human HR+ breast cancer cell lines to palbociclib for 7–9 months. In vitro mechanistic study and in vivo xenograft assay were performed. For clinical relevance, public mRNA microarray data sets of early breast cancer were analyzed and PEG10 immunohistochemical staining was performed using pre-CDK4/6 inhibitor tumor samples.ResultsWe observed that PEG10 was significantly upregulated in palbociclib-resistant cells. Ectopic overexpression of PEG10 in parental cells caused CDK4/6 inhibitor resistance and enhanced epithelial–mesenchymal transition (EMT). On the contrary, PEG10-targeting siRNA or antisense oligonucleotides (ASOs) combined with palbociclib synergistically inhibited proliferation of palbociclib-resistant cells and growth of palbociclib-resistant xenograft in mice and suppressed EMT as well. The mechanistic study confirmed that high PEG10 expression suppressed p21, a natural CDK inhibitor, and SIAH1, a post-translational degrader of ZEB1, augmenting CDK4/6 inhibitor resistance. Then PEG10 siRNA combined with palbociclib suppressed cell cycle progression and EMT via activating p21 and SIAH1, respectively. Consequently, combined PEG10 inhibition and palbociclib overcame CDK4/6 inhibitor resistance. Furthermore, high PEG10 expression was significantly associated with a shorter recurrence-free survival (RFS) based on public mRNA expression data. In pre-CDK4/6 inhibitor treatment tissues, PEG10 positivity by IHC also showed a trend toward a shorter progression-free survival (PFS) with CDK4/6 inhibitor. These results support clinical relevance of PEG10 as a therapeutic target.ConclusionsWe demonstrated a novel PEG10-associated mechanism of CDK4/6 inhibitor resistance. We propose PEG10 as a promising therapeutic target for overcoming PEG10-associated resistance to CDK4/6 inhibitors.

Simple SummaryCyclin-dependent kinases 4 and 6 (CDK4/6) are key enzymes controlling the cell cycle. CDK4/6 inhibitors are being tested in multiple clinical trials for a range of cancers including melanoma, and a deeper understanding of how they interact with other therapies is vital for their clinical development. Beyond the cell cycle, CDK4/6 regulates cell metabolism, which is a critical factor determining response to standard-of-care mitogen-activated protein kinase (MAPK) pathway therapies in melanoma. Here, we show that CDK4/6 inhibitors increase glutamine and fatty acid-oxidation-dependent mitochondrial metabolism in melanoma cells, but they do not alter the metabolic response to MAPK inhibitors. These observations shed light on how CDK4/6 inhibitors impinge on the regulation of metabolism and how they interact with other therapies in the setting of melanoma.Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are being tested in numerous clinical trials and are currently employed successfully in the clinic for the treatment of breast cancers. Understanding their mechanism of action and interaction with other therapies is vital in their clinical development. CDK4/6 regulate the cell cycle via phosphorylation and inhibition of the tumour suppressor RB, and in addition can phosphorylate many cellular proteins and modulate numerous cellular functions including cell metabolism. Metabolic reprogramming is observed in melanoma following standard-of-care BRAF/MEK inhibition and is involved in both therapeutic response and resistance. In preclinical models, CDK4/6 inhibitors overcome BRAF/MEK inhibitor resistance, leading to sustained tumour regression; however, the metabolic response to this combination has not been explored. Here, we investigate how CDK4/6 inhibition reprograms metabolism and if this alters metabolic reprogramming observed upon BRAF/MEK inhibition. Although CDK4/6 inhibition has no substantial effect on the metabolic phenotype following BRAF/MEK targeted therapy in melanoma, CDK4/6 inhibition alone significantly enhances mitochondrial metabolism. The increase in mitochondrial metabolism in melanoma cells following CDK4/6 inhibition is fuelled in part by both glutamine metabolism and fatty acid oxidation pathways and is partially dependent on p53. Collectively, our findings identify new p53-dependent metabolic vulnerabilities that may be targeted to improve response to CDK4/6 inhibitors.

Radiosensitizing Effects of CDK4/6 Inhibitors in Hormone Receptor-Positive and HER2-Negative Breast Cancer are through Downregulating DNA Repair Mechanism and NF-κB-Signaling Pathway

Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We examined levels of 172 phosphorylated and non-phosphorylated proteins under conditions of Ionizing radiation (IR) in patient derived GBM stem cells and established U251, U87 GBM cell lines in vitro and in an in vivo orthotropic mouse model. We identified subsets of proteins with clearly concordant/discordant behavior between GBM cells in vitro and in vivo. In general, molecules involved in anti-apoptotic, cell-cycle, survival pathways, tumor metastasis and DNA repair were affected. Comparing in vivo and in vitro samples after IR, 9 proteins were commonly elevated; phospho(p)-STAT3, CDC2, CyclinB1, BAX, pEIF4BP1, pAKT, pRB, pMEK1, and FOXM1. Conversely, 4 other proteins were commonly decreased; pPRKCA, pPRKCD, pNDRG1 and pRPS6. Recent evidence of FOXM1 as a master regulator of metastasis and its important role in maintaining neural, progenitor, and GBM stem cells intrigued us to validate it as a radiosensitizing target. We show high expression of FOXM1 across different patient derived stem cells. When GBM stem cells (NSC11, GBAM1) were differentiated in serum, we observed a decrease in FOXM1 levels, attaining more differentiation markers. In both differentiated and un-differentiated GBM stem cells, treatment with IR resulted in an increase of FOXM1 expression. However, inhibition of FOXM1 was only seen to have an effect on un-differentiated GBM stem cells, and resulted in reduced cell viability, a significant reduction in clonogenicity, and anchorage-independent growth, along with enhanced radiosenstivity with IR. Importantly, the combination of IR with FOXM1 inhibition showed these same effects irrespective of serum-differentiation. These results clearly suggest, inhibition of FOXM1 leads to radiosensitization. Since GBM stem cells, which comprise a subpopulation of tumor cells, maybe responsible for therapeutic resistance, we show that FOXM1 inhibition stands as a potential cancer stem-cell specific chemo-radio therapeutic target for GBM. Citation Format: Uday Bhanu Maachani, Anita T. Tandle, Uma Shankavaram, Tamalee Meushaw, Philip J. Tofilon, Kevin A. Camphausen. Profiling signaling networks using reverse phase protein arrays: validating FOXM1 as a potential target to radiosensitize glioblastoma (GBM) stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 849. doi:10.1158/1538-7445.AM2014-849

Aim: Cyclin-dependent kinases 4 and 6 (CDK4/6) are frequently upregulated in pancreatic ductal adenocarcinoma (PDAC) and are associated with poor overall survival. Although CDK4/6 inhibition suppresses tumor cell proliferation, it paradoxically promotes metastasis and invasion, and the mechanisms underlying this effect remain unclear.Methods: We evaluated the effects of the CDK4/6 inhibitor palbociclib (PD-0332991) and the bromodomain and extra-terminal (BET) inhibitor JQ1, administered individually and in combination, on human PDAC cell lines in vitro and on tumor growth in an orthotopic mouse model.Results: Palbociclib modestly inhibited pancreatic tumor growth but significantly enhanced tumor cell migration, invasion, and epithelial-to-mesenchymal transition (EMT). In contrast, co-treatment with JQ1 potentiated palbociclib’s anti-proliferative effects and reversed EMT. Mechanistically, CDK4/6 inhibition activated the canonical Wnt/β-catenin pathway via Ser9 phosphorylation of GSK3β, whereas BET inhibition disrupted the cross-talk between Wnt/β-catenin and TGF-β/Smad signaling. Combined inhibition of CDK4/6 and BET produced a synergistic antitumor effect in vitro and in vivo.Conclusion: Our findings support a combined therapeutic strategy targeting CDK4/6 and BET proteins to achieve synergistic inhibition of PDAC progression.

Phosphorylation of the retinoblastoma protein (Rb) by cyclin-dependent kinases 4 and 6 (CDK4/6) is a critical checkpoint for G1/S cell cycle progression and commitment to cellular proliferation. Human malignancies often subvert these control mechanisms through a range of genetic and biochemical adaptations. Accordingly, tumors that depend on CDK4/6 activity for proliferation and survival are particularly sensitive to inhibition of this pathway by palbociclib (IbranceTM), a highly selective inhibitor of CDK4/6 kinase activities. Treatment regimen of palbociclib with letrozole significantly improved progression-free survival in a randomized phase 2 study of women with advanced estrogen receptor-positive (ER+), HER2-negative breast cancer. Likewise, in ER+ breast cancer models palbociclib and estrogen antagonists combine for greater anti-proliferative activity, increased hallmarks of cellular senescence and prolonged durability of response following drug removal. Dual inhibition of CDK4/6 and ER signaling demonstrated robust anti-tumor activity in xenograft studies. The addition of Palbociclib to other targeted therapeutics elicits improved activity in pre-clinical models of several non-breast indications and these effects also manifest through modulation of cellular proliferation, senescence and growth arrest. Data will be presented on the molecular basis of combination benefit with Palbociclib in ER+ breast and other oncology indications. Citation Format: Stephen Dann, Jing Yuan, John Chionis, Chaoting Liu, Tao Xie, Nathan V. Lee, Enhong Chen, Ping Wei, Paul A. Rejto, David J. Shields, Todd VanArsdale. Mechanistic basis of Palbociclib combinatorial activity in ER+ breast cancer and non-breast indications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2740.

The Novel Multitarget Small-Molecule Inhibitor SRX3177 Overcomes Ibrutinib Resistance in Mantle Cell Lymphoma

We conducted a phase Ib study (NCT02345824) to determine whether ribociclib, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), penetrates tumor tissue and modulates downstream signaling pathways including retinoblastoma protein (Rb) in patients with recurrent glioblastoma (GBM). Study participants received ribociclib (600mg QD) for 8-21days before surgical resection of their recurrent GBM. Total and unbound concentrations of ribociclib were measured in samples of tumor tissue, plasma, and cerebrospinal fluid (CSF). We analyzed tumor specimens obtained from the first (initial/pre-study) and second (recurrent/on-study) surgery by immunohistochemistry for Rb status and downstream signaling of CDK4/6 inhibition. Participants with Rb-positive recurrent tumors continued ribociclib treatment on a 21-day-on, 7-day-off schedule after surgery, and were monitored for toxicity and disease progression. Three participants with recurrent Rb-positive GBM participated in this study. Mean unbound (pharmacologically active) ribociclib concentrations in plasma, CSF, MRI-enhancing, MRI-non-enhancing, and tumor core regions were 0.337μM, 0.632μM, 1.242nmol/g, 0.484nmol/g, and 1.526nmol/g, respectively, which exceeded the in vitro IC50 (0.04μM) for inhibition of CDK4/6 in cell-free assay. Modulation of pharmacodynamic markers of ribociclib CDK 4/6 inhibition in tumor tissues were inconsistent between study participants. No participants experienced serious adverse events, but all experienced early disease progression. This study suggests that ribociclib penetrated recurrent GBM tissue at concentrations predicted to be therapeutically beneficial. Our study was unable to demonstrate tumor pharmacodynamic correlates of drug activity. Although well tolerated, ribociclib monotherapy seemed ineffective for the treatment of recurrent GBM.