Jeffry L. Dean

Therapeutic CDK4/6 inhibition in breast cancer: key mechanisms of response and failure.

A hallmark of cancer is the deregulation of cell-cycle machinery, ultimately facilitating aberrant proliferation that fuels tumorigenesis and disease progression. Particularly, in breast cancers, cyclin D1 has a crucial role in the development of disease. Recently, a highly specific inhibitor of CDK4/6 activity (PD-0332991) has been developed that may have efficacy in the treatment of breast cancer. To interrogate the utility of PD-0332991 in treating breast cancers, therapeutic response was evaluated on a panel of breast cancer cell lines. These analyses showed that the chronic loss of Rb is specifically associated with evolution to a CDK4/6-independent state and, ultimately, resistance to PD-0332991. However, to interrogate the functional consequence of Rb directly, knockdown experiments were performed in models that represent immortalized mammary epithelia and multiple subtypes of breast cancer. These studies showed a highly specific role for Rb in mediating the response to CDK4/6 inhibition that was dependent on transcriptional repression manifest through E2F, and the ability to attenuate CDK2 activity. Acquired resistance to PD-03322991 was specifically associated with attenuation of CDK2 inhibitors, indicating that redundancy in CDK functions represents a determinant of therapeutic failure. Despite these caveats, in specific models, PD-0332991 was a particularly effective therapy, which induced Rb-dependent cytostasis. Combined, these findings indicate the critical importance of fully understanding cell-cycle regulatory pathways in directing the utilization of CDK inhibitors in the clinic.

Dual roles of PARP-1 promote cancer growth and progression

UNLABELLED PARP-1 is an abundant nuclear enzyme that modifies substrates by poly(ADP-ribose)-ylation. PARP-1 has well-described functions in DNA damage repair and also functions as a context-specific regulator of transcription factors. With multiple models, data show that PARP-1 elicits protumorigenic effects in androgen receptor (AR)-positive prostate cancer cells, in both the presence and absence of genotoxic insult. Mechanistically, PARP-1 is recruited to sites of AR function, therein promoting AR occupancy and AR function. It was further confirmed in genetically defined systems that PARP-1 supports AR transcriptional function, and that in models of advanced prostate cancer, PARP-1 enzymatic activity is enhanced, further linking PARP-1 to AR activity and disease progression. In vivo analyses show that PARP-1 activity is required for AR function in xenograft tumors, as well as tumor cell growth in vivo and generation and maintenance of castration resistance. Finally, in a novel explant system of primary human tumors, targeting PARP-1 potently suppresses tumor cell proliferation. Collectively, these studies identify novel functions of PARP-1 in promoting disease progression, and ultimately suggest that the dual functions of PARP-1 can be targeted in human prostate cancer to suppress tumor growth and progression to castration resistance. SIGNIFICANCE These studies introduce a paradigm shift with regard to PARP-1 function in human malignancy, and suggest that the dual functions of PARP-1 in DNA damage repair and transcription factor regulation can be leveraged to suppress pathways critical for promalignant phenotypes in prostate cancer cells by modulation of the DNA damage response and hormone signaling pathways. The combined studies highlight the importance of dual PARP-1 function in malignancy and provide the basis for therapeutic targeting.

Therapeutically activating RB: reestablishing cell cycle control in endocrine therapy-resistant breast cancer

The majority of estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy. While this is effective, acquired resistance to therapies targeted against ER is a major clinical challenge. Here, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These analyses revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. In this context, cyclin D1 expression and retinoblastoma tumor suppressor protein (RB) phosphorylation are maintained even with efficient ablation of ER with pure antagonists. These therapy-resistant models recapitulate a key feature of deregulated RB/E2F transcriptional control. Correspondingly, a gene expression signature of RB-dysfunction is associated with luminal B breast cancer, which exhibits a relatively poor response to endocrine therapy. 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, a highly selective CDK4/6 inhibitor, PD-0332991, was effective at suppressing the proliferation of all hormone refractory models analyzed. Importantly, PD-0332991 led to a stable cell cycle arrest that was fundamentally distinct from those elicited by ER antagonists, and was capable of inducing aspects of cellular senescence in hormone therapy refractory cell populations. These findings underscore the clinical utility of downstream cytostatic therapies in treating tumors that have experienced failure of endocrine therapy.

A Hormone–DNA Repair Circuit Governs the Response to Genotoxic Insult

UNLABELLED Alterations in DNA repair promote tumor development, but the impact on tumor progression is poorly understood. Here, discovery of a biochemical circuit linking hormone signaling to DNA repair and therapeutic resistance is reported. Findings show that androgen receptor (AR) activity is induced by DNA damage and promotes expression and activation of a gene expression program governing DNA repair. Subsequent investigation revealed that activated AR promotes resolution of double-strand breaks and resistance to DNA damage both in vitro and in vivo. Mechanistically, DNA-dependent protein kinase catalytic subunit (DNAPKcs) was identified as a key target of AR after damage, controlling AR-mediated DNA repair and cell survival after genotoxic insult. Finally, DNAPKcs was shown to potentiate AR function, consistent with a dual role in both DNA repair and transcriptional regulation. Combined, these studies identify the AR-DNAPKcs circuit as a major effector of DNA repair and therapeutic resistance and establish a new node for therapeutic intervention in advanced disease. SIGNIFICANCE The present study identifies for the fi rst time a positive feedback circuit linking hormone action to the DNA damage response and shows the significant impact of this process on tumor progression and therapeutic response. These provocative findings provide the foundation for development of novel nodes of therapeutic intervention for advanced disease.

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.

RB-pathway disruption in breast cancer: differential association with disease subtypes, disease-specific prognosis and therapeutic response.

In breast cancer, inactivation of the RB tumor suppressor gene is believed to occur via multiple mechanisms to facilitate tumorigenesis. However, the prognostic and predictive value of RB status in disease-specific clinical outcomes has remained uncertain. We investigated RB pathway deregulation in the context of both ER-positive and ER-negative disease using combined microarray datasets encompassing over 900 breast cancer patient samples. Disease-specific characteristics of RB pathway deregulation were investigated in this dataset by evaluating correlation among pathway genes as well as differential expression across patient tumor populations defined by ER status. Survival analysis among these breast cancer samples demonstrates that the RB-loss signature is associated with poor disease outcome within several independent cohorts. Within the ER-negative subpopulation, the RB-loss signature is associated with improved response to chemotherapy and longer relapse-free survival. Additionally, while individual genes in the RB target signature closely reproduce its prognostic value, they also serve to predict and monitor response to therapeutic compounds, such as the cytostatic agent PD-0332991. These results indicate that the RB-loss signature expression is associated with poor outcome in breast cancer, but predicts improved response to chemotherapy based on data in ER-negative populations. While the RB-loss signature, as a whole, demonstrates prognostic and predictive utility, a small subset of markers could be sufficient to stratify patients based on RB function and inform the selection of appropriate therapeutic regimens.

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.

Cyclin D1b protein expression in breast cancer is independent of cyclin D1a and associated with poor disease outcome.

Aberrant expression of cyclin D1 protein is a common feature of breast cancer. However, the CCND1 gene encodes two gene products, cyclin D1a and cyclin D1b, which have discrete mechanisms of regulation and impact on cell behavior. A polymorphism at nucleotide 870 in the CCND1 gene, rs603965, influences the relative production of the encoded proteins and can impart increased risk for tumor development. Here, the impact of both the G/A870 polymorphism and cyclin D1b protein production on breast cancer risk, disease phenotype and patient outcome was analysed. In a large multiethnic case–control study, the G/A870 polymorphism conferred no significant risk for breast cancer overall or by stage or estrogen receptor (ER) status. However, the cyclin D1b protein was found to be upregulated in breast cancer, independent of cyclin D1a levels, and exhibited heterogeneous levels in breast cancer specimens. High cyclin D1a expression inversely correlated with the Ki67 proliferation marker and was not associated with clinical outcome. In contrast, elevated cyclin D1b expression was independently associated with adverse outcomes, including recurrence, distant metastasis and decreased survival. Interestingly, cyclin D1b was particularly associated with poor outcome in the context of ER-negative breast cancer. Thus, specific cyclin D1 isoforms are associated with discrete forms of breast cancer and high cyclin D1b protein levels hold prognostic potential.

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.

USP22 regulates oncogenic signaling pathways to drive lethal cancer progression.

Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease.