Sheena M. Daignault

Cell Cycle Phase-Specific Drug Resistance as an Escape Mechanism of Melanoma Cells

The tumor microenvironment is characterized by cancer cell subpopulations with heterogeneous cell cycle profiles. For example, hypoxic tumor zones contain clusters of cancer cells that arrest in G1 phase. It is conceivable that neoplastic cells exhibit differential drug sensitivity based on their residence in specific cell cycle phases. In this study, we used two-dimensional and organotypic melanoma culture models in combination with fluorescent cell cycle indicators to investigate the effects of cell cycle phases on clinically used drugs. We demonstrate that G1-arrested melanoma cells, irrespective of the underlying cause mediating G1 arrest, are resistant to apoptosis induced by the proteasome inhibitor bortezomib or the alkylating agent temozolomide. In contrast, G1-arrested cells were more sensitive to mitogen-activated protein kinase pathway inhibitor-induced cell death. Of clinical relevance, pretreatment of melanoma cells with a mitogen-activated protein kinase pathway inhibitor, which induced G1 arrest, resulted in resistance to temozolomide or bortezomib. On the other hand, pretreatment with temozolomide, which induced G2 arrest, did not result in resistance to mitogen-activated protein kinase pathway inhibitors. In summary, we established a model to study the effects of the cell cycle on drug sensitivity. Cell cycle phase-specific drug resistance is an escape mechanism of melanoma cells that has implications on the choice and timing of drug combination therapies.

Abstract 314: Cell cycle phase-specific drug resistance as an escape mechanism of melanoma cells

The tumor microenvironment is characterized by cancer cell subpopulations with heterogeneous cell cycle profiles. For example, hypoxic areas within tumors contain clusters of cancer cells that arrest in the G1 cell cycle phase. It is conceivable that cancer cells exhibit differential drug sensitivity based on their residence in specific cell cycle phases. Here, we have used two-dimensional and organotypic melanoma culture models in combination with fluorescent cell cycle indicators to investigate the effects of G1-arrest on clinically used melanoma drugs. We demonstrate that G1-arrested melanoma cells, irrespective of the underlying cause mediating G1-arrest, are resistant to induced cell death by the proteasome inhibitor bortezomib and the alkylating agent temozolomide. In contrast, G1-arrested cells were more sensitive to MAPK pathway inhibition. Of clinical relevance, pre-treatment of melanoma cells with a MAPK pathway inhibitor resulted in resistance to temozolomide or bortezomib, while in contrast pre-treatment with temozolomide did not result in resistance to the MAPK pathway inhibitor. In summary, we have established a model to study the effects of the cell cycle on drug sensitivity. Cell cycle phase-specific drug resistance is an escape mechanism of melanoma cells that has implications on the choice and timing of drug combination therapies. Citation Format: Kimberley A. Beaumont, David S. Hill, Sheena M. Daignault, Danae M. Sharp, Brian Gabrielli, Wolfgang Weninger, Nikolas K. Haass. Cell cycle phase-specific drug resistance as an escape mechanism of melanoma cells. [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 314.

Endogenous replication stress marks melanomas sensitive to CHK1 inhibitors in vivo

Purpose: Checkpoint kinase 1 inhibitors (CHEK1i) have single-agent activity in vitro and in vivo. Here, we have investigated the molecular basis of this activity. Experimental Design: We have assessed a panel of melanoma cell lines for their sensitivity to the CHEK1i GNE-323 and GDC-0575 in vitro and in vivo. The effects of these compounds on responses to DNA replication stress were analyzed in the hypersensitive cell lines. Results: A subset of melanoma cell lines is hypersensitive to CHEK1i-induced cell death in vitro, and the drug effectively inhibits tumor growth in vivo. In the hypersensitive cell lines, GNE-323 triggers cell death without cells entering mitosis. CHEK1i treatment triggers strong RPA2 hyperphosphorylation and increased DNA damage in only hypersensitive cells. The increased replication stress was associated with a defective S-phase cell-cycle checkpoint. The number and intensity of pRPA2 Ser4/8 foci in untreated tumors appeared to be a marker of elevated replication stress correlated with sensitivity to CHEK1i. Conclusions: CHEK1i have single-agent activity in a subset of melanomas with elevated endogenous replication stress. CHEK1i treatment strongly increased this replication stress and DNA damage, and this correlated with increased cell death. The level of endogenous replication is marked by the pRPA2Ser4/8 foci in the untreated tumors, and may be a useful marker of replication stress in vivo. Clin Cancer Res; 24(12); 2901–12. ©2018 AACR.

Abstract C189: Defective S phase cell cycle checkpoint: A potential culprit and target in melanoma

The S phase cell cycle checkpoint is critical for maintaining genomic integrity. This checkpoint is triggered in response to DNA damage and replication stresses to control cell cycle progression, halting DNA replication and allowing the time to repair to maintain genomic integrity. If this checkpoint is defective, cells lose their ability to ensure the fidelity of replication and repair any damage, resulting in replicative stress and genomic instability which will subsequently increase the risk of cancer. We have discovered that a large proportion of melanoma cell lines lose the ability to halt replication when challenged with high dose hydroxyurea-induced replicative stress, despite normal cell cycle checkpoint signalling. We found that failure in the crosstalk between ATR-CHK1 checkpoint signalling and the cell cycle mechanism in destabilization of CDC25A. Although CHK1-dependent phosphorylation of CDC25A which triggers its degradation was detected, the S phase checkpoint defective melanoma cell lines fail to destabilize CDC25A, maintaining normal activation of CDK2/Cyclin E. The melanoma cells with this S phase checkpoint defect are also hyper-sensitive to killing by CHK1 inhibitor. We have previously shown that melanoma with high levels of replicative stress are very sensitive to CHK1 inhibitors. I will report our studies investigating whether CHK1 inhibitor sensitivity is a direct outcome of the replicative stress arising from the S phase checkpoint defect, or whether the loss of the ATR-CHK1-CDC25A dependent S phase checkpoint signalling mechanism is directly responsible for the hyper-sensitivity to CHK1 inhibitors. Citation Format: Zay Y. Oo, Sheena Daignault, James Chen, Brian Gabrielli. Defective S phase cell cycle checkpoint: A potential culprit and target in melanoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C189.

Melanoma tumor sub-populations are defined by MITF expression, and exhibit enhanced proliferation and characteristics of an EMT

HUMMER, DOUGLAS ANDREW. Community College Presidential Change from the Department Leader’s Perspective: A Case Study. (Under the direction of Dr. Diane Chapman). Academic department leaders are the least studied level of management in higher education, yet they play a fundamental role in transforming the vision and goals of executive leadership into reality (Gonaim, 2016). Academic department leaders play such a role because as a group, they influence the largest part of the employee population at a college; the faculty (Stringer, 2002; Tierney, 1999). Since the ability to create lasting change is one of the core qualities of a highly effective community college president (The Aspen Institute & Achieving the Dream, 2013), it is important to understand how the strategies, tactics, and actions employed by the executive leadership of an institution of higher learning shape the perceptions academic department leaders have of their workplace. This study took a qualitative approach to understanding these perceptions during the first few months of new president’s administration at a large community college in the southeast US. A transcendental phenomenological (Husserl, 2004) lens and case study methodologies were used to collect and analyze structural and textural data to describe the perceptions and interpretations academic department leaders had of this presidential change phenomenon. The case study itself provided the structural description, or context of the phenomenon, and semi-structured interviews were used to give voice to the academic department leaders that participated in the study. An analysis of the data revealed a number of themes that addressed the research questions asked in this study. These themes led to the following findings in this study. Listening sessions initiated by the new president and the hiring process that brought him to the college were identified as major influencing events in creating a positive impression of this new executive and his vision for the future of the institution. This positive impression is a contributor to creating an organizational climate ready to accept change. Other themes that emerged revolved around executive leadership’s support, communication issues, the hierarchical levels of management, changes that were initiated, and the fear to speak up. The findings from this study can be used by many community colleges as they hire new presidents and prepare for the changes new executives bring. The findings can also be used by any college approaching a major change initiative. The presidential change phenomenon will affect as many as four out of every five community colleges over the next 10 years (Phillippe, 2016). Presidential changes and other major change initiatives that community colleges experience will provide many opportunities to repeat this study and share the findings with others experiencing the same phenomenon.

Melanoma cells in G1 phase escape proteasome inhibitor cytotoxicity

HUMMER, DOUGLAS ANDREW. Community College Presidential Change from the Department Leader’s Perspective: A Case Study. (Under the direction of Dr. Diane Chapman). Academic department leaders are the least studied level of management in higher education, yet they play a fundamental role in transforming the vision and goals of executive leadership into reality (Gonaim, 2016). Academic department leaders play such a role because as a group, they influence the largest part of the employee population at a college; the faculty (Stringer, 2002; Tierney, 1999). Since the ability to create lasting change is one of the core qualities of a highly effective community college president (The Aspen Institute & Achieving the Dream, 2013), it is important to understand how the strategies, tactics, and actions employed by the executive leadership of an institution of higher learning shape the perceptions academic department leaders have of their workplace. This study took a qualitative approach to understanding these perceptions during the first few months of new president’s administration at a large community college in the southeast US. A transcendental phenomenological (Husserl, 2004) lens and case study methodologies were used to collect and analyze structural and textural data to describe the perceptions and interpretations academic department leaders had of this presidential change phenomenon. The case study itself provided the structural description, or context of the phenomenon, and semi-structured interviews were used to give voice to the academic department leaders that participated in the study. An analysis of the data revealed a number of themes that addressed the research questions asked in this study. These themes led to the following findings in this study. Listening sessions initiated by the new president and the hiring process that brought him to the college were identified as major influencing events in creating a positive impression of this new executive and his vision for the future of the institution. This positive impression is a contributor to creating an organizational climate ready to accept change. Other themes that emerged revolved around executive leadership’s support, communication issues, the hierarchical levels of management, changes that were initiated, and the fear to speak up. The findings from this study can be used by many community colleges as they hire new presidents and prepare for the changes new executives bring. The findings can also be used by any college approaching a major change initiative. The presidential change phenomenon will affect as many as four out of every five community colleges over the next 10 years (Phillippe, 2016). Presidential changes and other major change initiatives that community colleges experience will provide many opportunities to repeat this study and share the findings with others experiencing the same phenomenon.

Abstract 1781: G1 phase melanoma cells escape proteasome inhibitor cytotoxicity

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Utilising the fluorescent ubiquitination-based cell cycle indicator (FUCCI), which facilitates real-time cell cycle tracking, we have demonstrated in vitro and in vivo that melanomas are composed of differentially cycling tumour cells in a subcompartment-specific distribution. Further, we have shown that targeting the endoplasmic reticulum with fenretinide (synthetic retinoid) or bortezomib (26S proteasome inhibitor) induces cell cycle arrest and apoptosis of metastatic melanoma cells in vitro and in vivo. This study aims to investigate the effect of ER stress-inducing agents on the dynamics of cell division and cell death of individual melanoma cells within the complex tumor microenvironment, and to develop combination strategies that increase the efficacy of ER stress-inducing agents for melanoma therapy. FUCCI-melanoma cells were grown as 3D spheroids and implanted into a collagen matrix to mimic tumor architecture and microenvironment. Utilising the F-XBP1ΔDBD-venus reporter construct, which labels the cytoplasm in response to ER stress, we found that bortezomib induced ER stress, delayed cell cycle progression, and combination with fenretinide increased cell death in 2D and 3D culture. Flow cytometry and confocal microscopy indicated that treatment of FUCCI-melanoma cells with bortezomib induced G2 accumulation in 2D and 3D culture over the course of 24 h. In contrast, by 72 h the majority of cells were in G1 phase. Analysis of 2D and 3D real-time cell cycle imaging movies revealed that bortezomib induced both G1- and G2 arrest, but preferentially killed G2-phase cells. Consequently, pretreatment with temozolomide or fenretinide, leading to G2-arrest, sensitised melanoma cells to bortezomib cytotoxicity. In contrast, pretreatment with MEK inhibitors, leading to G1 arrest, inhibited bortezomib cytotoxicity in all melanoma cells, as did selective BRAF inhibitors in BRAF mutant melanoma cells. Our data suggest that bortezomib combined with fenretinide or temozolamide is a strategy worth exploring for the treatment of BRAF-inhibitor insensitive or resistant melanoma. Importantly, melanoma cells arrested in G1 are protected from bortezomib cytotoxicity, which excludes MAPK pathway inhibitors as combination partners. Citation Format: Sheena M. Daignault, David S. Hill, Kimberley A. Beaumont, Andrea Anfosso, Penny E. Lovat, Wolfgang Weninger, Nikolas K. Haass. G1 phase melanoma cells escape proteasome inhibitor cytotoxicity. [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 1781. doi:10.1158/1538-7445.AM2015-1781

MITF regulates proliferative subpopulation tumor architecture and modifies invasion and characteristics of the epithelial to mesenchymal transition within melanoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Melanoma is the leading cause of skin cancer-related death. Survival rates are high if the disease is diagnosed early, but drop precipitously at later stages. Small molecule inhibitor therapy has given robust responses in the clinic, but relapse is almost certain. This relapse may be due, in part, to tumor heterogeneity. Not only are there multiple cell types within a tumor, but cancer cells themselves can exhibit various phenotypes. This can be due to genotype variation or nutrient availability, and result in populations with different proliferative and invasive capabilities. As these cells display various behaviors, they could also respond to therapies uniquely. Understanding the molecular signature influencing different sub-populations is therefore crucial to design the most effective therapeutic regimen. The fluorescence ubiquitination cell cycle indicator (FUCCI) system, which delineates phases of the cell cycle by visual means, was employed to better understand melanoma tumor heterogeneity. Using this model, it was found that tumor xenografts grown in mice produce two cohorts. One that contained distinct clusters of either arrested or proliferating cells, and another that displayed a homogenous dispersion of proliferating cells throughout the breadth of the tumor. These cohorts were subsequently discovered to display either low or high levels of microphthalmia-associated transcription factor (MITF) expression, respectively. Additionally, loss of MITF by shRNA treatment resulted in conversion of the ability of melanoma cells to give rise to a homogenous xenograft, to instead produce a clustered tumor phenotype. Furthermore, in a 3D in vitro tumor spheroid model, MITF expression was predominantly found in the periphery of the spheroid, which corresponds with the region of highly proliferative cells. Forced over-expression of MITF within these spheroids results in loss of the distinct proliferative ring, and instead a homogenous growth pattern. Not only do spheroids express MITF around the perimeter, but also markers of the Epithelial to Mesenchymal Transition (EMT). These markers, such as Vimentin and Slug, also switch to become expressed homogenously upon high MITF expression. Surprisingly, the increased levels of EMT marker expression by MITF do not correlate to increased migration, and these spheroids in fact show reduced invasion into collagen. We are currently exploring what other means of cancer cell migration could trump an enhanced EMT phenotype and slow invasion in our model. These data outline how tumor heterogeneity, including proliferative and invasive potential, is tightly intertwined with MITF expression, making it an important marker for therapy design. Citation Format: Crystal A. Tonnessen, Kimberley A. Beaumont, David S. Hill, Sheena M. Daignault, Andrea Anfosso, Russell J. Jurek, Wolfgang Weninger, Nikolas K. Haass. MITF regulates proliferative subpopulation tumor architecture and modifies invasion and characteristics of the epithelial to mesenchymal transition within melanoma. [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 1420. doi:10.1158/1538-7445.AM2015-1420