A. Kathleen McClendon

Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors

To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.

Modification of the DNA Damage Response by Therapeutic CDK4/6 Inhibition

Background: Targeted CDK4/6 inhibition is a novel therapeutic strategy undergoing PhaseI/II clinical trials for the treatment of solid tumors. Results: CDK4/6 inhibition antagonizes the cytotoxic mechanism(s) of traditional chemotherapies and alters DNA repair processes. Conclusion: CDK4/6 inhibition attenuates the cellular response to cytotoxic chemotherapies. Significance: Understanding of cell cycle and transcriptional effects of CDK4/6 inhibition is critical for clinical utilization. The RB/E2F axis represents a critical node of cell signaling that integrates a diverse array of signaling pathways. Recent evidence has suggested a role for E2F-mediated gene transcription in DNA damage response and repair, as well as apoptosis signaling. Herein, we investigated how repression of E2F activity via CDK4/6 inhibition and RB activation impacts the response of triple negative breast cancer (TNBC) to frequently used therapeutic agents. In combination with taxanes and anthracyclines CDK4/6 inhibition and consequent cell cycle arrest prevented the induction of DNA damage and associated cell death in an RB-dependent manner; thereby demonstrating antagonism between the cytostatic influence of the CDK-inhibitor and cytotoxic agents. As many of these effects were secondary to cell cycle arrest, γ-irradiation (IR) was utilized to examine effects of CDK4/6 inhibition on direct DNA damage. Although E2F controls a number of genes involved in DNA repair (e.g. Rad51), CDK4/6 inhibition did not alter the overall rate of DNA repair, rather it significantly shifted the burden of this repair from homologous recombination (HR) to non-homologous end joining (NHEJ). Together, these data indicate that CDK4/6 inhibition can antagonize cytotoxic therapeutic strategies and increases utilization of error-prone DNA repair mechanisms that could contribute to disease progression.

RB and p53 Cooperate to Prevent Liver Tumorigenesis in Response to Tissue Damage

BACKGROUND & AIMS The tumor suppressors retinoblastoma (RB) and p53 are important regulators of the cell cycle. Although human cancer cells inactivate RB and p53 by many mechanisms, the cooperative roles of these proteins in tumorigenesis are complex and tissue specific. We analyzed the cooperation of RB and p53 in liver development and pathogenesis of hepatocellular carcinoma. METHODS Spontaneous and carcinogen-induced (diethylnitrosamine) tumorigenesis were studied in mice with liver-specific deletions of Rb and/or p53 (Rbf/f;albcre+, p53f/f;albcre+ and Rbf/f; p53f/f;albcre+ mice). Genotype, histologic, immunohistochemical, microarray, quantitative polymerase chain reaction, immunoblot, and comparative genomic hybridization analyses were performed using normal and tumor samples. Comparative microarray analyses were performed against publicly available human microarray data sets. RESULTS Deletion of RB and p53 from livers of mice deregulated the transcriptional programs associated with human disease. These changes were not sufficient for spontaneous tumorigenesis; potent quiescence mechanisms compensated for loss of these tumor suppressors. In response to hepatocarcinogen-induced damage, distinct and cooperative roles of RB and p53 were revealed; their loss affected cell cycle control, checkpoint response, and genome stability. In damaged tissue, combined loss of RB and p53 resulted in early lesion formation, aggressive tumor progression, and gene expression signatures and histologic characteristics of advanced human hepatocellular carcinoma. CONCLUSIONS The effects RB and p53 loss are determined by the tissue environment; cell stresses that promote aggressive disease reveal the functions of these tumor suppressors.

Retinoblastoma and Phosphate and Tensin Homolog Tumor Suppressors: Impact on Ductal Carcinoma In Situ Progression

BACKGROUND A subset of patients with ductal carcinoma in situ (DCIS) will progress to invasive breast cancer. However, there are currently no markers to differentiate women at high risk from those at lower risk of developing invasive disease. METHODS The association of two major tumor suppressor genes, retinoblastoma (RB) and phosphatase and tensin homolog (PTEN), with risk of any ipsilateral breast event (IBE) or progression to invasive breast cancer (IBC) was analyzed using data from 236 DCIS patients treated with breast conserving surgery with long-term follow-up. RB and PTEN expression was assessed with immunohistochemistry. The functional effects of RB and/or PTEN loss were modeled in MCF10A cells. Hazard ratios (HRs) were estimated with univariate and multivariable Cox regression models. All statistical tests were two-sided. RESULTS Loss of RB immunoreactivity in DCIS was strongly associated with risk of IBE occurrence (HR = 2.64; 95% confidence interval [CI] = 1.64 to 4.25) and IBC recurrence (HR = 4.66; 95% CI = 2.19 to 9.93). The prognostic power of RB loss remained statistically significant in multivariable analyses. PTEN loss occurred frequently in DCIS but was not associated with recurrence or progression. However, patients with DCIS lesions that were both RB and PTEN deficient were at further increased risk for IBEs (HR = 3.39; 95% CI = 1.92 to 5.99) and IBC recurrence (HR = 6.1, 95% CI = 2.5 to 14.76). Preclinical modeling in MCF10A cells demonstrated that loss of RB and PTEN impacted proliferation, motility, and invasive properties. CONCLUSIONS These studies indicate that RB and PTEN together have prognostic utility and could be used to target aggressive treatment for patients with the greatest probability of benefit.

RB loss contributes to aggressive tumor phenotypes in MYC-driven triple negative breast cancer

Triple negative breast cancer (TNBC) is characterized by multiple genetic events occurring in concert to drive pathogenic features of the disease. Here we interrogated the coordinate impact of p53, RB, and MYC in a genetic model of TNBC, in parallel with the analysis of clinical specimens. Primary mouse mammary epithelial cells (mMEC) with defined genetic features were used to delineate the combined action of RB and/or p53 in the genesis of TNBC. In this context, the deletion of either RB or p53 alone and in combination increased the proliferation of mMEC; however, the cells did not have the capacity to invade in matrigel. Gene expression profiling revealed that loss of each tumor suppressor has effects related to proliferation, but RB loss in particular leads to alterations in gene expression associated with the epithelial-to-mesenchymal transition. The overexpression of MYC in combination with p53 loss or combined RB/p53 loss drove rapid cell growth. While the effects of MYC overexpression had a dominant impact on gene expression, loss of RB further enhanced the deregulation of a gene expression signature associated with invasion. Specific RB loss lead to enhanced invasion in boyden chambers assays and gave rise to tumors with minimal epithelial characteristics relative to RB-proficient models. Therapeutic screening revealed that RB-deficient cells were particularly resistant to agents targeting PI3K and MEK pathway. Consistent with the aggressive behavior of the preclinical models of MYC overexpression and RB loss, human TNBC tumors that express high levels of MYC and are devoid of RB have a particularly poor outcome. Together these results underscore the potency of tumor suppressor pathways in specifying the biology of breast cancer. Further, they demonstrate that MYC overexpression in concert with RB can promote a particularly aggressive form of TNBC.

Unique Impact of RB Loss on Hepatic Proliferation TUMORIGENIC STRESSES UNCOVER DISTINCT PATHWAYS OF CELL CYCLE CONTROL

The retinoblastoma (RB) tumor suppressor pathway is disrupted at high frequency in hepatocellular carcinoma. However, the mechanisms through which RB modulates physiological responses in the liver remain poorly defined. Despite the well established role of RB in cell cycle control, the deletion of RB had no impact on the kinetics of cell cycle entry or the restoration of quiescence during the course of liver regeneration. Although these findings indicated compensatory effects from the RB-related proteins p107 and p130, even the dual deletion of RB with p107 or p130 failed to deregulate hepatic proliferation. Furthermore, although these findings suggested a modest role for the RB-pathway in the context of proliferative control, RB loss had striking effects on response to the genotoxic hepatocarcinogen diethylnitrosamine. With diethylnitrosamine, RB deletion resulted in inappropriate cell cycle entry that facilitated secondary genetic damage and further uncoupling of DNA replication with mitotic entry. Analysis of the mechanism underlying the differential impact of RB status on liver biology revealed that, while liver regeneration is associated with the conventional induction of cyclin D1 expression, the RB-dependent cell cycle entry, occurring with diethylnitrosamine treatment, was independent of cyclin D1 levels and associated with the specific induction of E2F1. Combined, these studies demonstrate that RB loss has disparate effects on the response to unique tumorigenic stresses, which is reflective of distinct mechanisms of cell cycle entry.

Differential impact of tumor suppressor pathways on DNA damage response and therapy-induced transformation in a mouse primary cell model.

The RB and p53 tumor suppressors are mediators of DNA damage response, and compound inactivation of RB and p53 is a common occurrence in human cancers. Surprisingly, their cooperation in DNA damage signaling in relation to tumorigenesis and therapeutic response remains enigmatic. In the context of individuals with heritable retinoblastoma, there is a predilection for secondary tumor development, which has been associated with the use of radiation-therapy to treat the primary tumor. Furthermore, while germline mutations of the p53 gene are critical drivers for cancer predisposition syndromes, it is postulated that extrinsic stresses play a major role in promoting varying tumor spectrums and disease severities. In light of these studies, we examined the tumor suppressor functions of these proteins when challenged by exposure to therapeutic stress. To examine the cooperation of RB and p53 in tumorigenesis, and in response to therapy-induced DNA damage, a combination of genetic deletion and dominant negative strategies was employed. Results indicate that loss/inactivation of RB and p53 is not sufficient for cellular transformation. However, these proteins played distinct roles in response to therapy-induced DNA damage and subsequent tumorigenesis. Specifically, RB status was critical for cellular response to damage and senescence, irrespective of p53 function. Loss of RB resulted in a dramatic evolution of gene expression as a result of alterations in epigenetic programming. Critically, the observed changes in gene expression have been specifically associated with tumorigenesis, and RB-deficient, recurred cells displayed oncogenic characteristics, as well as increased resistance to subsequent challenge with discrete therapeutic agents. Taken together, these findings indicate that tumor suppressor functions of RB and p53 are particularly manifest when challenged by cellular stress. In the face of such challenge, RB is a critical suppressor of tumorigenesis beyond p53, and RB-deficiency could promote significant cellular evolution, ultimately contributing to a more aggressive disease.

Abstract 5375: Reestablishing cell cycle control in breast cancers through therapeutic CDK4/6 inhibition

One hallmark of cancer is the deregulation of cell cycle machinery, which ultimately serves to promote aberrant proliferation that fuels tumorigenesis and progression. Particularly, the cyclin D1/Rb/E2F axis plays a crucial role in the development of disease by serving to connect aberrant mitogenic signaling with cell cycle transcriptional programs. In this context, the role of estrogen receptor (ER) in breast cancers has been previously described to exert influence upstream of this axis. While first-line strategies targeting ER activity are prevalent and initially effective in the majority of ER-positive breast cancers, acquired resistance and therapeutic failure remain a major clinical challenge. Current analyses have revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. As a gene expression signature of cyclin D/Rb/E2F-dysfunction was found to be associated with luminal B breast cancers (which are known to exhibit a relatively poor response to endocrine therapy), the prevalence of ER dysfunction occurring upstream of cyclin D1 suggests that targeting CDK 4/6 activity may be effective. To this end, we examined the therapeutic response to a CDK 4/6 inhibitor (PD-0332991) in human breast cancer cell lines and demonstrated a highly-specific role for Rb in mediating this response, which was dependent on transcriptional repression manifest through E2F activity. These analyses additionally demonstrated that the chronic loss of Rb can promote evolution to a CDK4/6-independent state and near complete resistance to PD-0332991. In addition to these studies, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These collective findings suggest that suppression of cyclin D-supported kinase activity and restoration of Rb-mediated transcriptional repression could represent a viable therapeutic option in tumors that fail to respond to hormone-based therapies. Consistent with this hypothesis, CDK4/6 inhibiton was effective at suppressing the proliferation of all hormone refractory models analyzed. Combined, these findings underscore the clinical utility of mediating aberrant cell cycle progression through down-stream cytostatic therapies in treating tumors that have experienced failure of first line and endocrine-based therapy, and provide rationale for incorporation of CDK 4/6 inhibition into current existing therapeutic regimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5375. doi:10.1158/1538-7445.AM2011-5375

Abstract 2982: Impact of the RB-pathway in breast cancer heterogeneity and therapeutic response

Molecular heterogeneity in breast cancer is associated with differential prognosis and response to therapy. However, in spite of extensive molecular classification, pathways that can be specifically targeted to understand the basis of disease and improve treatment remain poorly understood within specific subtypes of breast cancer. The retinoblastoma tumor suppressor (RB) pathway is deregulated in most cancers; however varied mechanisms of inactivation have significant impacts on tumor biology. Here novel mechanistic and preclinical models were combined with analyses of tumor specimens to decipher the differential impact of the RB-pathway within breast cancer subtypes and as a specific target for patient stratification and therapeutic targeting. Initial gene expression datasets encompassing over 2000 breast cancer patient samples were used to demonstrate a striking dichotomy in the mechanisms of RB functional inactivation within subtypes of disease. In ER-positive breast cancer, disruption of RB-pathway function was associated with poor prognosis and failure of endocrine therapy. Functional studies demonstrate that such therapeutic failure largely occurs in the presence of an intact Rb gene, as a result of deregulated mitogenic signaling. In such models of hormone refractory disease we demonstrate that pharmacological activation of RB is a potent second-line therapy. In contrast, in ER-negative tumor populations RB inactivation occurs in 40-50% of cases. This event yields complete loss of RB protein, as occurs through genetic and epigenetic mechanisms. Strikingly, in ER-negative disease RB loss is associated with improved response to chemotherapy and longer relapse-free survival. The basis for improved response involves both deregulation of cell cycle checkpoints and apoptotic processes. Ongoing analyses are using synthetic-lethal and combination-therapy approaches to rationally target RB-status in the management of breast cancer. In total, these studies define a clear role for RB-pathway as a determinant of tumor heterogeneity in breast cancer that can be leveraged to more effectively treat breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2982. doi:10.1158/1538-7445.AM2011-2982