Smruthi Vijayaraghavan

CDK4/6 and autophagy inhibitors synergistically induce senescence in Rb positive cytoplasmic cyclin E negative cancers

Deregulation of the cell cycle machinery is a hallmark of cancer. While CDK4/6 inhibitors are FDA approved (palbociclib) for treating advanced estrogen receptor-positive breast cancer, two major clinical challenges remain: (i) adverse events leading to therapy discontinuation and (ii) lack of reliable biomarkers. Here we report that breast cancer cells activate autophagy in response to palbociclib, and that the combination of autophagy and CDK4/6 inhibitors induces irreversible growth inhibition and senescence in vitro, and diminishes growth of cell line and patient-derived xenograft tumours in vivo. Furthermore, intact G1/S transition (Rb-positive and low-molecular-weight isoform of cyclin E (cytoplasmic)-negative) is a reliable prognostic biomarker in ER positive breast cancer patients, and predictive of preclinical sensitivity to this drug combination. Inhibition of CDK4/6 and autophagy is also synergistic in other solid cancers with an intact G1/S checkpoint, providing a novel and promising biomarker-driven combination therapeutic strategy to treat breast and other solid tumours.

AXL Inhibition Suppresses the DNA Damage Response and Sensitizes Cells to PARP Inhibition in Multiple Cancers

Epithelial to mesenchymal transition (EMT) is associated with a wide range of changes in cancer cells, including stemness, chemo- and radio-resistance, and metastasis. The mechanistic role of upstream mediators of EMT has not yet been well characterized. Recently, we showed that non–small cell lung cancers (NSCLC) that have undergone EMT overexpress AXL, a receptor tyrosine kinase. AXL is also overexpressed in a subset of triple-negative breast cancers (TNBC) and head and neck squamous cell carcinomas (HNSCC), and its overexpression has been associated with more aggressive tumor behavior and linked to resistance to chemotherapy, radiotherapy, and targeted therapy. Because the DNA repair pathway is also altered in patient tumor specimens overexpressing AXL, it is hypothesized that modulation of AXL in cells that have undergone EMT will sensitize them to agents targeting the DNA repair pathway. Downregulation or inhibition of AXL directly reversed the EMT phenotype, led to decreased expression of DNA repair genes, and diminished efficiency of homologous recombination (HR) and RAD51 foci formation. As a result, AXL inhibition caused a state of HR deficiency in the cells, making them sensitive to inhibition of the DNA repair protein, PARP1. AXL inhibition synergized with PARP inhibition, leading to apoptotic cell death. AXL expression also associated positively with markers of DNA repair across TNBC, HNSCC, and NSCLC patient cohorts. Implications: The novel role for AXL in DNA repair, linking it to EMT, suggests that AXL can be an effective therapeutic target in combination with targeted therapy such as PARP inhibitors in several different malignancies. Mol Cancer Res; 15(1); 45–58. ©2016 AACR.

Sequential Combination Therapy of CDK Inhibition and Doxorubicin Is Synthetically Lethal in p53-Mutant Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive malignancy in which the tumors lack expression of estrogen receptor, progesterone receptor, and HER2. Hence, TNBC patients cannot benefit from clinically available targeted therapies and rely on chemotherapy and surgery for treatment. While initially responding to chemotherapy, TNBC patients are at increased risk of developing distant metastasis and have decreased overall survival compared with non-TNBC patients. A majority of TNBC tumors carry p53 mutations, enabling them to bypass the G1 checkpoint and complete the cell cycle even in the presence of DNA damage. Therefore, we hypothesized that TNBC cells are sensitive to cell-cycle–targeted combination therapy, which leaves nontransformed cells unharmed. Our findings demonstrate that sequential administration of the pan-CDK inhibitor roscovitine before doxorubicin treatment is synthetically lethal explicitly in TNBC cells. Roscovitine treatment arrests TNBC cells in the G2–M cell-cycle phase, priming them for DNA damage. Combination treatment increased frequency of DNA double-strand breaks, while simultaneously reducing recruitment of homologous recombination proteins compared with doxorubicin treatment alone. Furthermore, this combination therapy significantly reduced tumor volume and increased overall survival compared with single drug or concomitant treatment in xenograft studies. Examination of isogenic immortalized human mammary epithelial cells and isogenic tumor cell lines found that abolishment of the p53 pathway is required for combination-induced cytotoxicity, making p53 a putative predictor of response to therapy. By exploiting the specific biologic and molecular characteristics of TNBC tumors, this innovative therapy can greatly impact the treatment and care of TNBC patients. Mol Cancer Ther; 15(4); 593–607. ©2016 AACR.

CDK4/6 Inhibitors Sensitize Rb-positive Sarcoma Cells to Wee1 Kinase Inhibition through Reversible Cell-Cycle Arrest

Research into the biology of soft tissue sarcomas has uncovered very few effective treatment strategies that improve upon the current standard of care which usually involves surgery, radiation, and chemotherapy. Many patients with large (>5 cm), high-grade sarcomas develop recurrence, and at that point have limited treatment options available. One challenge is the heterogeneity of genetic drivers of sarcomas, and many of these are not validated targets. Even when such genes are tractable targets, the rarity of each subtype of sarcoma makes advances in research slow. Here we describe the development of a synergistic combination treatment strategy that may be applicable in both soft tissue sarcomas as well as sarcomas of bone that takes advantage of targeting the cell cycle. We show that Rb-positive cell lines treated with the CDK4/6 inhibitor palbociclib reversibly arrest in the G1 phase of the cell cycle, and upon drug removal cells progress through the cell cycle as expected within 6–24 hours. Using a long-term high-throughput assay that allows us to examine drugs in different sequences or concurrently, we found that palbociclib-induced cell-cycle arrest poises Rb-positive sarcoma cells (SK-LMS1 and HT-1080) to be more sensitive to agents that work preferentially in S–G2 phase such as doxorubicin and Wee1 kinase inhibitors (AZD1775). The synergy between palbociclib and AZD1775 was also validated in vivo using SK-LMS1 xenografts as well as Rb-positive patient-derived xenografts (PDX) developed from leiomyosarcoma patients. This work provides the necessary preclinical data in support of a clinical trial utilizing this treatment strategy. Mol Cancer Ther; 16(9); 1751–64. ©2017 AACR.

Molecular genetics and cellular events of K-Ras-driven tumorigenesis

Cellular transformation and the accumulation of genomic instability are the two key events required for tumorigenesis. K-Ras (Kirsten-rat sarcoma viral oncogene homolog) is a prominent oncogene that has been proven to drive tumorigenesis. K-Ras also modulates numerous genetic regulatory mechanisms and forms a large tumorigenesis network. In this review, we track the genetic aspects of K-Ras signaling networks and assemble the sequence of cellular events that constitute the tumorigenesis process, such as regulation of K-Ras expression (which is influenced by miRNA, small nucleolar RNA and lncRNA), activation of K-Ras (mutations), generation of reactive oxygen species (ROS), induction of DNA damage and apoptosis, induction of DNA damage repair pathways and ROS detoxification systems, cellular transformation after apoptosis by the blebbishield emergency program and the accumulation of genomic/chromosomal instability that leads to tumorigenesis.

Inhibiting CDK in Cancer Therapy: Current Evidence and Future Directions

Cell cycle dysregulation is a hallmark of all cancers, resulting in uncontrolled proliferation. Cyclin dependent kinases (CDKs), a family of proteins that are involved in the regulation of the cell cycle, are frequently overexpressed or mutated in cancer. Hence, CDK-inhibiting drugs have been developed and evaluated as cancer therapeutics. Clinical trials have shown CDK4/6 inhibitors (CDK4/6i) to be relatively safe and effective, and these are now standard of care treatment for advanced hormone receptor positive breast cancer. Some CDK4/6i drugs are also able to cross the blood brain barrier and may, therefore, offer effective therapy for primary and metastatic central nervous system malignancies. Ongoing research is also evaluating CDK4/6i for additional breast cancer subtypes and non-breast malignancies with promising early phase clinical trial results. Finally, pre-clinical research has identified potential biomarkers for CDK4/6i efficacy and is exploring potential resistance mechanisms to this treatment. Further clinical-translational research is needed to advance patient selection and combinatorial treatment strategies with CDK4/6i in breast cancer and other malignancies.

Synthetic lethality of PARP inhibitors in combination with MYC blockade is independent of BRCA status in triple negative breast cancer

PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients. Several of those patients exhibit intrinsic/acquired resistance mechanisms that limit efficacy of PARPi monotherapy. Here we show how the efficacy of PARPi in triple-negative breast cancers (TNBC) can be expanded by targeting MYC-induced oncogenic addiction. In BRCA-mutant/sporadic TNBC patients, amplification of the MYC gene is correlated with increased expression of the homologous DNA recombination enzyme RAD51 and tumors overexpressing both genes are associated with worse overall survival. Combining MYC blockade with PARPi yielded synthetic lethality in MYC-driven TNBC cells. Using the cyclin-dependent kinase inhibitor dinaciclib, which downregulates MYC expression, we found that combination with the PARPi niraparib increased DNA damage and downregulated homologous recombination, leading to subsequent downregulation of the epithelial-mesenchymal transition and cancer stem-like cell phenotypes. Notably, dinaciclib resensitized TBNC cells, which had acquired resistance to niraparib. We found that the synthetic lethal strategy employing dinaciclib and niraparib was also highly efficacious in ovarian, prostate, pancreatic, colon, and lung cancer cells. Taken together, our results show how blunting MYC oncogene addiction can leverage cancer cell sensitivity to PARPi, facilitating the clinical use of c-myc as a predictive biomarker for this treatment.Significance: Dual targeting of MYC-regulated homologous recombination and PARP-mediated DNA repair yields potent synthetic lethality in triple-negative breast tumors and other aggressive tumors characterized by MYC overexpression. Cancer Res; 78(3); 742-57. ©2017 AACR.

Abstract 1783: Pharmacological inhibition of CDK4/6 induces G1 arrest, autophagy and senescence in ER+ breast cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Deregulation of the cell cycle machinery is a hallmark of most cancers, leading to aberrant proliferation and tumorigenesis, making cell cycle proteins, particularly cyclin dependent kinases (CDKs), attractive and druggable targets. Palbociclib or PD0332991, a potent CDK4/6 inhibitor, is an anti-proliferative agent that induces G1 arrest and prevents tumor growth in breast cancer. This drug has shown tremendous success in phase II clinical trials in ER+ breast cancer and phase III trials are underway. Despite promising results in the clinic, little is known about palbociclibs mechanism of action or modes of resistance in ER+ breast cancer. A clear understanding of these mechanisms are critical to identify biomarkers and understand the biology of treatment response, drug resistance and combination strategies, and this project is aimed at addressing these gaps in knowledge. We have used ER+ breast cancer cell lines (MCF7, T47D) to examine the mechanism of action of palbociclib and identify nodes that mediate drug resistance. Our results revealed that treatment with palbociclib induces a G1 arrest with concomitant downregulation of phospho-Rb. Furthermore, treated cells undergo autophagy and senescence in a dose-dependent manner. The autophagic response observed is a pro-survival mechanism and pharmacological inhibition of autophagy augments the cells sensitivity to palbociclib. Since this agent is known to be a specific CDK4/6 inhibitor, we downregulated these kinases in our model system and subjected them to drug treatment. Interestingly, knockdown of CDK4 or CDK6 did not completely recapitulate the drugs anti-tumor effects, suggesting that palbociclib could inhibit another target, albeit, at a higher concentration. We then interrogated if the Rb-FOXM1 pathway was sufficient to mediate palbociclib-induced growth inhibition in ER+ breast cancer. Results from drug-response studies revealed that knockdown of Rb reduces the sensitivity of ER+ cells by 4-6 fold, but the role of FOXM1 remains to be understood. Lastly, to interrogate the modes of acquired resistance, we developed palbocilcib-resistant cell lines, wherein we observed an overexpression of oncogenic low molecular weight isoforms of cyclin-E (LMW-E). Additionally, drug-response studies revealed that LMW-E overexpression can mediate resistance to palbociclib, indicating that LMW-E could serve as a biomarker of resistance. Collectively, our studies provide a rationale for utilizing palbociclib in the clinic in combination with an inhibitor of autophagy, such as hydroxychloroquine. Further, our results suggest that inhibition of G1/S transition is not the sole mode of action of palbociclib and that there could be another unidentified target that is inhibited, when the G1/S checkpoint is compromised. Identification of this target will be instrumental to overcome resistance and determine biomarkers of sensitivity to this agent in breast cancer patients. Citation Format: Smruthi Vijayaraghavan, Khandan Keyomarsi. Pharmacological inhibition of CDK4/6 induces G1 arrest, autophagy and senescence in ER+ breast cancer. [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 1783. doi:10.1158/1538-7445.AM2015-1783

Abstract 2060: Characterizing acquired resistance to palbociclib in breast cancer

The CDK4/6 inhibitor palbociclib is currently being used in combination with endocrine therapy to treat advanced ER positive breast cancer patients. While this treatment has shown great promise in the clinic, about 25% of the patients do not respond, and almost all patients eventually acquire resistance to palbociclib treatment. Hence, understanding the mechanism(s) of acquired resistance to CDK4/6 inhibition is crucial to devise alternate treatment strategies. To interrogate this, we developed MCF7 and T47D resistant cells by treating them with increasing doses of palbociclib over a 6-month period. After confirming that these cells were resistant to palbociclib, we performed genome-wide expression analysis via RNA-seq, in comparison with the parental (sensitive) cells. RNA- seq analysis revealed 2888 differentially expressed genes (p Citation Format: Smruthi Vijayaraghavan, Iman Doostan, Jason P.W. Carey, Khandan Keyomarsi. Characterizing acquired resistance to palbociclib in breast cancer [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 2060. doi:10.1158/1538-7445.AM2017-2060

Abstract 2338: CDK4/6 and autophagy inhibitors synergize to induce senescence in cancers with an intact G1/S checkpoint

Deregulation of the cell cycle machinery is a hallmark of most cancers. The crucial role of the CDK4/6-CyclinD pathway in tumorigenesis has led to the successful development and FDA approval (palbociclib) of CDK4/6 inhibitors for the treatment of advanced estrogen receptor positive breast cancer. However, two major clinical challenges remain: i) lack of a reliable biomarker to predict treatment response and ii) adverse events leading to interruption or discontinuation of treatment which possibly thus curtailing therapeutic benefit. We found that treatment of ER+ breast cancer cell lines with the CDK4/6 inhibitor palbociclib resulted in a dose-dependent sustained growth inhibition and senescence. Interestingly, breast cancer cells activate autophagy in response to palbociclib, a stress response process that promotes cancer cell survival. Genetic ablation of crucial autophagic genes or pharmacological inhibition of autophagy increases the sensitivity of ER+ breast cancer cells to palbociclib and the other CDK4/6 inhibitors, ribociclib and abemaciclib. This was confirmed in vivo, where the combination of palbociclib and autophagy inhibitor, hydroxychloroquine (HCQ) resulted in a significantly improved and a sustained tumor shrinkage. To identify biomarkers that predict response to CDK4/6 inhibition, we examined the G1 checkpoint proteins and found that knockdown of Rb or overexpression of the oncogenic low molecular weight isoforms of Cyclin-E (LMW-E) mediates resistance to palbociclib and its combination with autophagy inhibitor. More significantly, immunohistochemically staining of pre-treatment biopsies from palbociclib treated patients strengthened the correlation between palbociclib efficacy and an intact G1/S checkpoint (Rb+ve /LMWE-ve), resulting in a significantly longer progression free survival compared to the other patient groups; thus solidifying Rb and LMW-E as reliable prognostic biomarkers for palbociclib treatment. Finally, we examined the biomarker driven synergy between CDK4/6 and autophagy inhibition in several other cancer cell lines. Several solid tumors (ovarian, lung, pancreatic, colon, prostate) and triple negative breast cancer (TNBC) cell lines exhibited a synergistic response to palbociclib/HCQ combination treatment dependent upon an intact G1/S transition (Rb+/LMWE-). This was also verified in a TNBC patient derived xenograft (PDX) model. Thus, this study addresses the aforementioned limitations and provides a novel and promising biomarker-driven combination therapeutic strategy to treat breast and other solid tumors. We predict that this combination of CDK4/6 and autophagy inhibitors would be more beneficial than standard dose palbociclib in patients, allowing us to lower the dose, minimize palbociclib mediated toxicities and potentially improve overall patient survival - a goal that has not yet been met with currently approved treatment combinations. Citation Format: Smruthi Vijayaraghavan, Cansu Karakas, Xian Chen, Iman Doostan, Akshara S. Raghavendra, Min Yi, Ravi Amaravadi, Kelly Hunt, Debu Tripathy, Khandan Keyomarsi. CDK4/6 and autophagy inhibitors synergize to induce senescence in cancers with an intact G1/S checkpoint [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 2338. doi:10.1158/1538-7445.AM2017-2338