Characterizing the contribution of the LXCXE binding cleft to pRB-mediated genome stability and tumor suppression

Condensation and segregation of mitotic chromosomes are critical processes for cellular propagation and if compromised, can lead to genomic instability. Genomic instability is known to be an active contributor to tumorigenesis, rather than being a by-product of malignant progression. The retinoblastoma protein (pRB) is the prototypic tumor suppressor. Its tumor suppressive properties are linked to its ability to negatively regulate proliferation by inhibiting E2F target gene transcription. Using a gene targeted mouse model defective for interactions mediated by the pRB LXCXE binding cleft that is distinct from E2F binding (Rb1ΔL/ΔL), I have demonstrated that LXCXE-interactions are an essential part of pRB-mediated tumor suppression. When these interactions are disrupted, cells exhibit chromosome condensation and mitotic defects that are unrelated to G1 to S-phase regulation by pRB. These defects contribute to earlier tumor formation and more aggressive pathology in Trp53+/- and Trp53-/- mouse models, revealing a new mechanism of tumor suppression, facilitated by pRB, whereby genome stability is maintained by the proper condensation of mitotic chromosomes. Subsequent study of the mechanism by which pRB facilitates genome stability suggests that a pRB-E2F1-Condensin II complex localizes to pericentromeric heterochromatin. In the absence of this complex, DNA double strand breaks are observed and persist into mitosis and the ensuing G1 phase of the cell division cycle. Moreover, haploinsufficiency of Rb1 was enough to compromise loading of Condensin II at pericentromeric DNA and elicit the same defects. Significantly, RB1+/- fibroblasts from retinoblastoma patients also exhibit DNA damage and mitotic errors. And, in cancers of mesenchymal origin, RB1+/- cells exhibit as much genomic instability as RB1-/- cells. Finally, haploinsufficiency of the LXCXE binding cleft of pRB compromises pRB-mediated tumor suppression, resulting in tumors with increased chromosome gains and losses, comparable to Rb1ΔL/ΔL mutant mice. The data presented in this thesis change our understanding of the importance of genome stability as a tumor suppressive mechanism of the retinoblastoma protein and contrary to traditional thought, suggests that haploinsufficiency of RB1 functionally contributes to tumorigenesis in humans

Disruption of CDK-resistant chromatin association by pRB causes DNA damage, mitotic errors, and reduces Condensin II recruitment

Organization of chromatin structure is indispensible to the maintenance of genome integrity. The retinoblastoma tumor suppressor protein (pRB) mediates both transcriptional repression and chromatin organization, but the independent contributions of these functions have been difficult to study. Here, we utilize a synthetic Rb1 mutant allele (F832A) that maintains pRB association at cell cycle gene promoters, but disrupts a cyclin-dependent kinase (CDK)-resistant interaction with E2F1 to reduce occupancy of pRB on intergenic chromatin. Reduced pRB chromatin association increases spontaneous γH2AX deposition and aneuploidy. Our data indicates that the CDK-resistant pRB-E2F1 scaffold recruits Condensin II to major satellite repeats to stabilize chromatin structure in interphase and mitosis through mechanisms that are distinct from silencing of repetitive sequence expression

Understanding the Attitudes and Beliefs of Oncologists Regarding the Transitioning and Sharing of Survivorship Care

Transitioning survivorship care from oncologists to primary care physicians (PCPs) is a reasonable alternative to oncologist-led care. This study assessed oncologists’ attitudes and beliefs regarding sharing/transitioning survivorship care. A prospective survey of oncologists within a regional cancer program assessing self-reported barriers and facilitators to sharing/transitioning survivorship care was disseminated. In total, 63% (n = 39) of surveyed oncologists responded. Patient preference (89%) and anxiety (84%) are key to transition of care decisions; reduced remuneration (95%) and fewer longitudinal relationships (63%) do not contribute. Oncologists agreed that more patients could be shared/transitioned. Barriers include treatment-related toxicities (82% agree), tumor-specific factors (60–90% agree) and perception of PCP willingness to participate in survivorship care (47% agree). Oncologists appear willing to share/transition more survivors to PCPs, though barriers exist that warrant further study. Understanding these issues is critical to developing policies supporting comprehensive survivorship care models that address both cancer and non-cancer health needs. The demonstrated feasibility of this project warrants a larger-scale survey of oncologists with respect to the transition of survivorship care to PCPs, to further inform effective interventions to support high-quality survivorship care

The Price of Success: Immune-Related Adverse Events from Immunotherapy in Lung Cancer

Cancer immunotherapy has the goal of enhancing a patient’s intrinsic immune processes in order to mount a successful immune response against tumor cells. Cancer cells actively employ tactics to evade, delay, alter, or attenuate the anti-tumor immune response. Immune checkpoint inhibitors (ICIs) modulate endogenous regulatory immune mechanisms to enhance immune system activation, and have become the mainstay of therapy in many cancer types. This activation occurs broadly and as a result, activation is supraphysiologic and relatively non-specific, which can lead to immune-related adverse events (irAEs), the frequency of which depends on the patient, the cancer type, and the specific ICI antibody. Careful assessment of patients for irAEs through history taking, physical exam, and routine laboratory assessments are key to identifying irAEs at early stages, when they can potentially be managed more easily and before progressing to higher grades or more serious effects. Generally, most patients with low grade irAEs are eligible for re-challenge with ICIs, and the use of corticosteroids to address an irAE is not associated with poorer patient outcomes. This paper reviews immune checkpoint inhibitors (ICIs) including their mechanisms of action, usage, associated irAEs, and their management

The Price of Success: Immune-Related Adverse Events from Immunotherapy in Lung Cancer

Cancer immunotherapy has the goal of enhancing a patient’s intrinsic immune processes in order to mount a successful immune response against tumor cells. Cancer cells actively employ tactics to evade, delay, alter, or attenuate the anti-tumor immune response. Immune checkpoint inhibitors (ICIs) modulate endogenous regulatory immune mechanisms to enhance immune system activation, and have become the mainstay of therapy in many cancer types. This activation occurs broadly and as a result, activation is supraphysiologic and relatively non-specific, which can lead to immune-related adverse events (irAEs), the frequency of which depends on the patient, the cancer type, and the specific ICI antibody. Careful assessment of patients for irAEs through history taking, physical exam, and routine laboratory assessments are key to identifying irAEs at early stages, when they can potentially be managed more easily and before progressing to higher grades or more serious effects. Generally, most patients with low grade irAEs are eligible for re-challenge with ICIs, and the use of corticosteroids to address an irAE is not associated with poorer patient outcomes. This paper reviews immune checkpoint inhibitors (ICIs) including their mechanisms of action, usage, associated irAEs, and their management

Understanding the Attitudes and Beliefs of Oncologists Regarding the Transitioning and Sharing of Survivorship Care

Transitioning survivorship care from oncologists to primary care physicians (PCPs) is a reasonable alternative to oncologist-led care. This study assessed oncologists’ attitudes and beliefs regarding sharing/transitioning survivorship care. A prospective survey of oncologists within a regional cancer program assessing self-reported barriers and facilitators to sharing/transitioning survivorship care was disseminated. In total, 63% (n = 39) of surveyed oncologists responded. Patient preference (89%) and anxiety (84%) are key to transition of care decisions; reduced remuneration (95%) and fewer longitudinal relationships (63%) do not contribute. Oncologists agreed that more patients could be shared/transitioned. Barriers include treatment-related toxicities (82% agree), tumor-specific factors (60–90% agree) and perception of PCP willingness to participate in survivorship care (47% agree). Oncologists appear willing to share/transition more survivors to PCPs, though barriers exist that warrant further study. Understanding these issues is critical to developing policies supporting comprehensive survivorship care models that address both cancer and non-cancer health needs. The demonstrated feasibility of this project warrants a larger-scale survey of oncologists with respect to the transition of survivorship care to PCPs, to further inform effective interventions to support high-quality survivorship care

Disruption of CDK-resistant chromatin association by pRB causes DNA damage, mitotic errors, and reduces Condensin II recruitment

Organization of chromatin structure is indispensible to the maintenance of genome integrity. The retinoblastoma tumor suppressor protein (pRB) mediates both transcriptional repression and chromatin organization, but the independent contributions of these functions have been difficult to study. Here, we utilize a synthetic Rb1 mutant allele (F832A) that maintains pRB association at cell cycle gene promoters, but disrupts a cyclin-dependent kinase (CDK)-resistant interaction with E2F1 to reduce occupancy of pRB on intergenic chromatin. Reduced pRB chromatin association increases spontaneous γH2AX deposition and aneuploidy. Our data indicates that the CDK-resistant pRB-E2F1 scaffold recruits Condensin II to major satellite repeats to stabilize chromatin structure in interphase and mitosis through mechanisms that are distinct from silencing of repetitive sequence expression

Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive

Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. In this report, we demonstrate that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, we studied this aspect of pRB function in isolation, and demonstrate that it is an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53−/− mice succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, we demonstrate that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53+/− mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes

A Functional Connection between pRB and Transforming Growth Factor β in Growth Inhibition and Mammary Gland Development▿

Transforming growth factor β (TGF-β) is a crucial mediator of breast development, and loss of TGF-β-induced growth arrest is a hallmark of breast cancer. TGF-β has been shown to inhibit cyclin-dependent kinase (CDK) activity, which leads to the accumulation of hypophosphorylated pRB. However, unlike other components of TGF-β cytostatic signaling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice carrying subtle missense changes in pRB (Rb1ΔL and Rb1NF), we have discovered that pRB plays a critical role in mammary gland development. In particular, Rb1 mutant female mice have hyperplastic mammary epithelium and defects in nursing due to insensitivity to TGF-β growth inhibition. In contrast with previous studies that highlighted the inhibition of cyclin/CDK activity by TGF-β signaling, our experiments revealed that active transcriptional repression of E2F target genes by pRB downstream of CDKs is also a key component of TGF-β cytostatic signaling. Taken together, our work demonstrates a unique functional connection between pRB and TGF-β in growth control and mammary gland development

Haploinsufficiency of an RB-E2F1-Condensin II Complex Leads to Aberrant Replication and Aneuploidy

Genome instability is a characteristic of malignant cells; however, evidence for its contribution to tumorigenesis has been enigmatic. In this study, we demonstrate that the retinoblastoma protein, E2F1, and Condensin II localize to discrete genomic locations including major satellite repeats at pericentromeres. In the absence of this complex, aberrant replication ensues followed by defective chromosome segregation in mitosis. Surprisingly, loss of even one copy of the retinoblastoma gene reduced recruitment of Condensin II to pericentromeres and caused this phenotype. Using cancer genome data and gene-targeted mice, we demonstrate that mutation of one copy of RB1 is associated with chromosome copy-number variation in cancer. Our study connects DNA replication and chromosome structure defects with aneuploidy through a dosage-sensitive complex at pericentromeric repeats. © 2014 American Association for Cancer Research