Eva Vidacs

Combined Targeting of JAK2 and Bcl-2/Bcl-xL to Cure Mutant JAK2-Driven Malignancies and Overcome Acquired Resistance to JAK2 Inhibitors

Summary To design rational therapies for JAK2-driven hematological malignancies, we functionally dissected the key survival pathways downstream of hyperactive JAK2. In tumors driven by mutant JAK2, Stat1, Stat3, Stat5, and the Pi3k and Mek/Erk pathways were constitutively active, and gene expression profiling of TEL-JAK2 T-ALL cells revealed the upregulation of prosurvival Bcl-2 family genes. Combining the Bcl-2/Bcl-xL inhibitor ABT-737 with JAK2 inhibitors mediated prolonged disease regressions and cures in mice bearing primary human and mouse JAK2 mutant tumors. Moreover, combined targeting of JAK2 and Bcl-2/Bcl-xL was able to circumvent and overcome acquired resistance to single-agent JAK2 inhibitor treatment. Thus, inhibiting the oncogenic JAK2 signaling network at two nodal points, at the initiating stage (JAK2) and the effector stage (Bcl-2/Bcl-xL), is highly effective and provides a clearly superior therapeutic benefit than targeting just one node. Therefore, we have defined a potentially curative treatment for hematological malignancies expressing constitutively active JAK2.

CDK9 inhibition by dinaciclib potently suppresses Mcl-1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo

MYC dysregulation confers a poor prognosis to diffuse large B-cell lymphoma (DLBCL), and effective therapeutic strategies are lacking in relapsed/refractory DLBCL, Burkitt lymphoma and intermediate forms.1, 2 As a master transcriptional regulator, MYC recruits transcription complexes containing RNA polymerase II (Pol II) to facilitate effective transcriptional elongation of MYC gene targets.3 Pol II is fully activated by phosphorylation of a critical serine residue at position 2 within heptapeptide repeats in the carboxy-terminal domain (CTD), a function performed by the positive transcription elongation factor b (P-TEFb; comprising CDK9 and cyclin T1).4 It has been shown that MYC binds and recruits P-TEFb to its targets as a means to activate Pol II.3, 5, 6 More recently, CDK9-mediated transcriptional elongation was reported as essential for tumor maintenance in a genetically defined MYC-driven model of hepatocellular carcinoma.7 Thus, CDK9 dependence may represent a druggable vulnerability in lymphomas with dysregulated MYC expression.

Preclinical screening of histone deacetylase inhibitors combined with ABT-737, rhTRAIL/MD5-1 or 5-azacytidine using syngeneic Vk*MYC multiple myeloma

Multiple myeloma (MM) is an incurable malignancy with an unmet need for innovative treatment options. Histone deacetylase inhibitors (HDACi) are a new class of anticancer agent that have demonstrated activity in hematological malignancies. Here, we investigated the efficacy and safety of HDACi (vorinostat, panobinostat, romidepsin) and novel combination therapies using in vitro human MM cell lines and in vivo preclinical screening utilizing syngeneic transplanted Vk*MYC MM. HDACi were combined with ABT-737, which targets the intrinsic apoptosis pathway, recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL/MD5-1), that activates the extrinsic apoptosis pathway or the DNA methyl transferase inhibitor 5-azacytidine. We demonstrate that in vitro cell line-based studies provide some insight into drug activity and combination therapies that synergistically kill MM cells; however, they do not always predict in vivo preclinical efficacy or toxicity. Importantly, utilizing transplanted Vk*MYC MM, we report that panobinostat and 5-azacytidine synergize to prolong the survival of tumor-bearing mice. In contrast, combined HDACi/rhTRAIL-based strategies, while efficacious, demonstrated on-target dose-limiting toxicities that precluded prolonged treatment. Taken together, our studies provide evidence that the transplanted Vk*MYC model of MM is a useful screening tool for anti-MM drugs and should aid in the prioritization of novel drug testing in the clinic.

The CDK9 inhibitor dinaciclib exerts potent apoptotic and antitumor effects in preclinical models of MLL-rearranged acute myeloid Leukemia

Translocations of the mixed lineage leukemia (MLL) gene occur in 60% to 80% of all infant acute leukemias and are markers of poor prognosis. MLL-AF9 and other MLL fusion proteins aberrantly recruit epigenetic regulatory proteins, including histone deacetylases (HDAC), histone methyltransferases, bromodomain-containing proteins, and transcription elongation factors to mediate chromatin remodeling and regulate tumorigenic gene expression programs. We conducted a small-molecule inhibitor screen to test the ability of candidate pharmacologic agents targeting epigenetic and transcriptional regulatory proteins to induce apoptosis in leukemic cells derived from genetically engineered mouse models of MLL-AF9-driven acute myeloid leukemia (AML). We found that the CDK inhibitor dinaciclib and HDAC inhibitor panobinostat were the most potent inducers of apoptosis in short-term in vitro assays. Treatment of MLL-rearranged leukemic cells with dinaciclib resulted in rapidly decreased expression of the prosurvival protein Mcl-1, and accordingly, overexpression of Mcl-1 protected AML cells from dinaciclib-induced apoptosis. Administration of dinaciclib to mice bearing MLL-AF9-driven human and mouse leukemias elicited potent antitumor responses and significantly prolonged survival. Collectively, these studies highlight a new therapeutic approach to potentially overcome the resistance of MLL-rearranged AML to conventional chemotherapies and prompt further clinical evaluation of CDK inhibitors in AML patients harboring MLL fusion proteins.

Functional-genetic dissection of HDAC dependencies in mouse lymphoid and myeloid malignancies.

Histone deacetylase (HDAC) inhibitors (HDACis) have demonstrated activity in hematological and solid malignancies. Vorinostat, romidepsin, belinostat, and panobinostat are Food and Drug Administration-approved for hematological malignancies and inhibit class II and/or class I HDACs, including HDAC1, 2, 3, and 6. We combined genetic and pharmacological approaches to investigate whether suppression of individual or multiple Hdacs phenocopied broad-acting HDACis in 3 genetically distinct leukemias and lymphomas. Individual Hdacs were depleted in murine acute myeloid leukemias (MLL-AF9;Nras(G12D); PML-RARα acute promyelocytic leukemia [APL] cells) and Eµ-Myc lymphoma in vitro and in vivo. Strikingly, Hdac3-depleted cells were selected against in competitive assays for all 3 tumor types. Decreased proliferation following Hdac3 knockdown was not prevented by BCL-2 overexpression, caspase inhibition, or knockout of Cdkn1a in Eµ-Myc lymphoma, and depletion of Hdac3 in vivo significantly reduced tumor burden. Interestingly, APL cells depleted of Hdac3 demonstrated a more differentiated phenotype. Consistent with these genetic studies, the HDAC3 inhibitor RGFP966 reduced proliferation of Eµ-Myc lymphoma and induced differentiation in APL. Genetic codepletion of Hdac1 with Hdac2 was pro-apoptotic in Eµ-Myc lymphoma in vitro and in vivo and was phenocopied by the HDAC1/2-specific agent RGFP233. This study demonstrates the importance of HDAC3 for the proliferation of leukemia and lymphoma cells, suggesting that HDAC3-selective inhibitors could prove useful for the treatment of hematological malignancies. Moreover, our results demonstrate that codepletion of Hdac1 with Hdac2 mediates a robust pro-apoptotic response. Our integrated genetic and pharmacological approach provides important insights into the individual or combinations of HDACs that could be prioritized for targeting in a range of hematological malignancies.

A community-based model of rapid autopsy in end-stage cancer patients

To the Editor: Systematic genomic studies, including the Cancer Genome Atlas (TCGA)1 and the International Cancer Genome Consortium (ICGC)2, have provided an unprecedented catalog of driver mutations in human cancer. However, these studies use mainly primary, pre-treatment tumor material obtained at surgery with curative intent. There is an urgent need to identify and characterize resistance mechanisms to understand how cancers can evade even the best medical efforts and kill patients; therefore, access to end-stage disease is important. Solid cancers show considerable spatial3, temporal4,5 and genomic heterogeneity at diagnosis. Selective pressure and mutagenic impact of treatment6 drives intra-patient evolution of cancer cell populations4,7. Understanding acquired resistance requires access to paired preand post-treatment samples4,7; however, curative surgery is typically confined to patients with locoregional disease, and opportunities for tumor sampling in advanced disseminated disease are limited. Here, we describe Cancer Tissue Collection After Death (CASCADE), an autopsy program that overcomes logistical challenges to enable collection of samples at end stage for research in melanoma and breast, ovarian and prostate cancers. For the CASCADE study, we aimed to recruit cancer patients close to the end of life, including those outside the minority of patients who die in hospitals. To preserve tissue integrity, autopsies must commence within a few hours of death, requiring access to around-the-clock services. Intervention in the emotionally charged end-of-life environment must be managed in an ethical manner and to a high standard. Finally, we aimed for the study to be highly cost-effective. We believe our approach to meeting these challenges is applicable to researchers in other large urban centers. Here we summarize the main steps in CASCADE’s operating protocol and our experiences from the initial 3 years and 30 autopsies performed (Fig. 1). Information about institutional review board approvals (including a detailed patient informationand-consent form), the autopsy procedure and certain laboratory processes is given in Supplementary Methods and Supplementary Figure 1. Recruitment of participants was led by the clinicians. Such discussions require careful consideration, in timing and in language, and were initiated only if there was a perception that tissue donation would be acceptable to the patients and their families. Factors suggesting acceptability include the emotional stability of the participant and family members and their clarity about and acceptance of the terminal nature of the disease. On occasion, participants prompted discussion by asking about organ or body donation. Consent discussions typically involved oncologists and/or palliative care physicians employed at recruiting hospitals who had established a care relationship with the participant and their family during the patient’s cancer journey. Frequently, the study was introduced at one meeting and discussed over several subsequent clinic visits, allowing patients and their families time to consider participation. We view the involvement of family members in the consent process as essential to support the participant and facilitate decisionmaking. Involvement of family members also ensures that they are fully aware of the autopsy process and helps to clarify funeral arrangements for the study team. After obtaining consent, study investigators collated clinical information, including that related to past and current treatment and diagnostic procedures such as imaging, on an ongoing basis. Between September 2012 and August 2015, 40 patients were approached, and 37 (92.5%) expressed interest in participating. Of those 32 patients (80%) consented; the other 5 had rapid clinical deterioration precluding

BET-inhibition induces apoptosis in aggressive B-cell lymphoma via epigenetic regulation of BCL-2 family members.

Targeting BET bromodomain proteins using small molecules is an emerging anticancer strategy with clinical evaluation of at least six inhibitors now underway. Although MYC downregulation was initially proposed as a key mechanistic property of BET inhibitors, recent evidence suggests that additional antitumor activities are important. Using the Eμ-Myc model of B-cell lymphoma, we demonstrate that BET inhibition with JQ1 is a potent inducer of p53-independent apoptosis that occurs in the absence of effects on Myc gene expression. JQ1 skews the expression of proapoptotic (Bim) and antiapoptotic (BCL-2/BCL-xL) BCL-2 family members to directly engage the mitochondrial apoptotic pathway. Consistent with this, Bim knockout or Bcl-2 overexpression inhibited apoptosis induction by JQ1. We identified lymphomas that were either intrinsically resistant to JQ1-mediated death or acquired resistance following in vivo exposure. Strikingly, in both instances BCL-2 was strongly upregulated and was concomitant with activation of RAS pathways. Eμ-Myc lymphomas engineered to express activated Nras upregulated BCL-2 and acquired a JQ1 resistance phenotype. These studies provide important information on mechanisms of apoptosis induction and resistance to BET-inhibition, while providing further rationale for the translation of BET inhibitors in aggressive B-cell lymphomas. Mol Cancer Ther; 15(9); 2030–41. ©2016 AACR.

Id2 and E proteins orchestrate the initiation and maintenance of MLL-rearranged acute myeloid leukemia

E proteins and their antagonists, the Id proteins, are transcriptional regulators important for normal hematopoiesis. We found that Id2 acts as a key regulator of leukemia stem cell (LSC) potential in MLL-rearranged acute myeloid leukemia (AML). Low endogenous Id2 expression is associated with LSC enrichment while Id2 overexpression impairs MLL-AF9-leukemia initiation and growth. Importantly, MLL-AF9 itself controls the E-protein pathway by suppressing Id2 while directly activating E2-2 expression, and E2-2 depletion phenocopies Id2 overexpression in MLL-AF9-AML cells. Remarkably, Id2 tumor-suppressive function is conserved in t(8;21) AML. Low expression of Id2 and its associated gene signature are associated with poor prognosis in MLL-rearranged and t(8;21) AML patients, identifying the Id2/E-protein axis as a promising new therapeutic target in AML.

A pharmacogenomic approach validates AG-221 as an effective and on-target therapy in IDH2 mutant AML

A pharmacogenomic approach validates AG-221 as an effective and on-target therapy in IDH2 mutant AML

Modulation of antitumour immune responses by intratumoural Stat1 expression

Signal transducer and activator of transcription 1 (Stat1) mediates anti‐viral responses and cytokine‐driven anti‐proliferative, apoptotic and immunomodulatory activities. As de‐regulated Stat1 function can affect tumour progression we sought to elucidate the effects of tumour cell‐intrinsic Stat1 expression on immunosurveillance. Knockout of Stat1 enhanced the development of sarcomas induced by the chemical carcinogen 3‐methylcholanthrene (MCA). Growth of transplanted MCA‐induced Stat1−/− sarcomas was suppressed in wild‐type mice compared to growth in Stat1−/− and immunocompromised recipients. Co‐depletion of NK and CD8+ T cells from wild‐type mice facilitated Stat1‐deficient tumour growth whereas depletion of CD4+ T cells and CD8+ T cells did not. In vitro and in vivo analysis of the tumours implicated a role for NK cell‐mediated, perforin‐dependent killing of Stat1‐deficient tumours. Interestingly, restoration of Stat1 expression in Stat1−/− tumours resulted in diminished involvement of NK cells and increased contribution of CD8+ T cells in anti‐tumour responses. Therefore, Stat1 expression within tumour cells modulated anti‐tumour immune responses by altering the dominant immune effector cell involvement from NK cells to CD8+ T cells in the absence or presence of Stat1 respectively.