Mary E. Klein

A model of sensitivity and resistance to histone deacetylase inhibitors in diffuse large B cell lymphoma: Role of cyclin-dependent kinase inhibitors

Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma. While the initial treatment strategy is highly effective, relapse occurs in 40% of cases. Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs but their single agent efficacy against relapsed DLBCL has been variable, ranging from few complete/partial responses to some stable disease. However, most patients showed no response to HDACi monotherapy for unknown reasons. Here we show that sensitivity and resistance to the hydroxamate HDACi, PXD101, can be modeled in DLBCL cell lines. Sensitivity is characterized by G2/M arrest and apoptosis and resistance by reversible G1 growth arrest. These responses to PXD101 are independent of several negative prognostic indicators such as DLBCL subtype, BCL2 and MYC co-expression, and p53 mutation, suggesting that HDACi might be used effectively against highly aggressive DLBCL tumors if they are combined with other therapeutics that overcome HDACi resistance. Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation. PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G1 arrest. The results suggest several treatment strategies that might increase the efficacy of HDACi against aggressive DLBCL.

ATRX is a regulator of therapy induced senescence in human cells

Senescence is a state of stable cell cycle exit with important implications for development and disease. Here, we demonstrate that the chromatin remodeling enzyme ATRX is required for therapy-induced senescence. ATRX accumulates in nuclear foci and is required for therapy-induced senescence in multiple types of transformed cells exposed to either DNA damaging agents or CDK4 inhibitors. Mobilization into foci depends on the ability of ATRX to interact with H3K9me3 histone and HP1. Foci form soon after cells exit the cell cycle, before other hallmarks of senescence appear. Eliminating ATRX in senescent cells destabilizes the senescence-associated heterochromatic foci. Additionally, ATRX binds to and suppresses expression from the HRAS locus; repression of HRAS is sufficient to promote the transition of quiescent cells into senescence and preventing repression blocks progression into senescence. Thus ATRX is a critical regulator of therapy-induced senescence and acts in multiple ways to drive cells into this state.Therapy induced senescence (TIS) is a growth suppressive program activated by cytostatic agents in some cancer cells. Here the authors show that the chromatin remodeling enzyme ATRX is a regulator of TIS and drives cells into this state via multiple mechanisms.

CDK4/6 Inhibitors: The Mechanism of Action May Not Be as Simple as Once Thought

CDK4/6 inhibitors are among a new generation of therapeutics. Building upon the striking success of the combination of CDK4/6 inhibitors and the hormone receptor antagonist letrozole in breast cancer, many other combinations have recently entered clinical trials in multiple diseases. To achieve maximal benefit with CDK4/6 inhibitors it will be critical to understand the cellular mechanisms by which they act. Here we highlight the mechanisms by which CDK4/6 inhibitors can exert their anti-tumor activities beyond simply enforcing cytostatic growth arrest, and discuss how this knowledge may inform new combinations, improve outcomes, and modify dosing schedules in the future.

Mechanistic understanding of the role of ATRX in senescence provides new insight for combinatorial therapies with CDK4 inhibitors.

ABSTRACT Senescence is an irreversible form of growth arrest and is generally considered a favorable outcome of cancer therapies, yet little is known about the molecular events that distinguish this state from readily reversible growth arrest (i.e. quiescence). Recently, we discovered that during therapy induced senescence the chromatin remodeling protein α-thalassemia, mental retardation, X-linked (ATRX) represses Harvey rat sarcoma viral oncogene homolog (HRAS), and repression of HRAS is necessary to establish senescence, suggesting how new clinical combinations might be used to achieve durable senescence.

PDLIM7 and CDH18 regulate the turnover of MDM2 during CDK4/6 inhibitor therapy-induced senescence

CDK4/6 inhibitors are being used to treat a variety of human malignancies. In well-differentiated and dedifferentiated liposarcoma their clinical promise is associated with their ability to downregulate the MDM2 protein. The downregulation of MDM2 following treatment with CDK4/6 inhibitors also induces many cultured tumor cell lines derived from different types of malignancies to progress from quiescence into senescence. Here we used cultured human cell lines and defined a role for PDLIM7 and CDH18, regulating MDM2 protein in CDK4/6 inhibitor-treated cells. Materials from our previous phase II trials with palbociclib were then used to demonstrate that expression of CDH18 protein was associated with response, measured as both progression-free survival and overall survival. This supports the hypothesis that the biologic transition from quiescence to senescence has clinical relevance for this class of drugs.

Abstract 597: The cellular and genomic response to histone deacetylase inhibitors in diffuse large b-cell lymphoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Diffuse Large B-cell lymphoma (DLBCL) is the most frequent subtype of Non-Hodgkin Lymphoma (NHL) in all countries around the world and in all age groups. Several drug regimens have been used in treatment of DLBCL; however, this disease remains eventually fatal in 30 – 40% of the patients. Chemotherapy resistance can be partly explained by the fact that DLBCL is a heterogeneous group of NHLs, with the two most prevalent subtypes being “Activated B-cell Like” (ABC) and “Germinal Center B-cell like” (GCB). Patients with the ABC subtype have the poorest prognosis under the current treatment regimen. Therefore, there is a pressing need for new therapeutics that can increase survival rates in DLBCL patients. Histone deacetylase inhibitors (HDIs) have proven to be promising drugs in the treatment of blood malignancies. Even though their mechanism of action has not been fully characterized, two HDIs, (Vorinostat and Romidepsin) have been approved for the treatment of cutaneous T-cell lymphoma (CTCL). Therefore the purpose of the current study is to investigate the response of DLBCL subtypes to HDIs, with a particular focus on subtype-specific mechanisms of action. Our current working hypothesis is that a comprehensive analysis of the genomic and proteomic response to histone deacetylase inhibitors (HDIs) including gene expression and transcription factor acetylation will reveal both mechanisms and potential biomarkers of HDI action in lymphomas DLBCL. In the current study we have focused on the cellular and genomic effects of the hydroxamate HDI, Belinostat (PXD101), on cell lines representing the GCB subtype of DLBCL. We show that PXD101 inhibits growth of four GCB-type cell lines with 24 h IC50s in the low micromolar range (SUDHL6 =0.15uM, OCI Ly19 = 0.3uM, SUDHL4 = 0.45uM, DB = 0.77uM). Flow cytometry analysis has shown that three of these cell lines (SUDHL6, OCI Ly19 and DB) arrest in the G2/M phase of the cell cycle by 24 hours of treatment at the IC50 dose and then die by apoptosis. In contrast, the SUDHL4 cell line reversibly arrests in the G1 phase without undergoing cell death. Western blot analysis of PARP and caspase-3 cleavage has further confirmed the presence/absence of apoptosis. We suggest that the SUDHL4 cell line represents DLBCL tumors that are refractory to the apoptosis-inducing effects of HDIs. Thus, we are using this cell line to identify other therapeutics which could be used in combination with PXD101 to induce cell death.The mechanistic basis for the differential cellular response between the GCB type cell lines is currently under investigation using expression profiling data obtained from OCI Ly19 and SUDHL4 cells treated with PXD101. Preliminary data indicates divergent responses in expression of GADD45 and p21, the Myc/Max family of proteins, and the clock genes, Per1 and Cry2. 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 597. doi:10.1158/1538-7445.AM2011-597