The mTORC1 inhibitor everolimus prevents and treats Eμ-Myc lymphoma by restoring oncogene-induced senescence

MYC deregulation is common in human cancer. IG-MYC translocations that are modeled in EμMyc mice occur in almost all cases of Burkitt lymphoma as well as in other B-cell lymphoproliferative disorders. Deregulated expression of MYC results in increased mTOR complex 1 (mTORC1) signaling. As tumors with mTORC1 activation are sensitive to mTORC1 inhibition, we used everolimus, a potent and specific mTORC1 inhibitor, to test the requirement for mTORC1 in the initiation and maintenance of EμMyc lymphoma. Everolimus selectively cleared premalignant B cells from the bone marrow and spleen, restored a normal pattern of B-cell differentiation, and strongly protected against lymphoma development. Established EμMyc lymphoma also regressed after everolimus therapy. Therapeutic response correlated with a cellular senescence phenotype and induction of p53 activity. Therefore, mTORC1-dependent evasion of senescence is critical for cellular transformation and tumor maintenance by MYC in B lymphocytes

Genomic characterisation of Eμ-Myc mouse lymphomas identifies Bcor as a Myc co-operative tumour-suppressor gene

The Eμ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eμ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eμ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.We acknowledge the following funding agencies: Leukaemia Foundation of Australia, Arrow Bone Marrow Transplant Foundation, National Health and Medical Research Council Australia, Cancer Council Victoria, Victorian Cancer Agency, Australian Cancer Research Foundation, Peter MacCallum Cancer Centre Foundation, National Institutes of Health

Targeting the phosphatidylinositol-3-kinase and mammalian target of rapamycin signalling pathway in MYC-driven models of B-cell neoplasia

© 2011 Dr. Jake ShorttThe phosphatidylinositol-3-kinase (PI3K) / mammalian target of rapamycin (mTOR) pathway links afferent signals from the microenvironment to the cellular machinery of growth, proliferation and survival. Aberrant PI3K activity is common in cancer, providing neoplastic cells with a competitive advantage over their normal counterparts. MYC is a prototypic oncogene that is deregulated in more than half of human cancers, but its activity is not readily amenable to pharmacological inhibition. Effectors of both PI3K signalling and MYC–regulated transcriptional networks intersect at multiple levels in cellular systems. This thesis examines interactions between PI3K/mTOR signalling and MYC, specifically exploring the therapeutic potential of PI3K/mTOR inhibition in MYC-driven haematological cancer. Eµ-Myc lymphoma is a tractable model of human Burkitt lymphoma. This thesis utilises genetic and pharmacological manipulation of the Eµ-Myc system to mechanistically define apoptosis induction by NVP-BEZ235, an ATP-competitive pan-PI3K/dual-mTOR inhibitor. By contrasting NVP-BEZ235 against more selective kinase inhibitors, we identify PI3K-related DNA damage response (DDR) kinases and mTORC-1 as key targets in this apoptotic response. NVP-BEZ235 induces p53-independent apoptosis at low nanomolar concentrations that inhibit mTORC-1 and DDR kinases, but are insufficient to further downregulate the low basal activity of PI3K and mTORC-2 in Eµ-Myc cells. Apoptosis correlates with biomarkers of stressed translational machinery and is dependent on a reciprocal induction of pro-apoptotic BMF and reductions in anti-apoptotic MCL-1. Similar activity of NVP-BEZ235 is observed in vivo, where drug treatment results in apoptotic lymphoma regressions and prolongation of survival. In vivo regressions correlate with inhibition of DDR-kinases and mTORC-1, but not antagonism of PI3K/AKT. However, genetic manipulation to activate AKT at the initiation of lymphomagenesis augments the response to NVP-BEZ235 in an ‘mTORC-1-addicted’ model of Myc-driven lymphoma. Here, NVP-BEZ235 and the allosteric mTORC-1 inhibitor, NVP-RAD001, induce curative responses. To broaden the scope of in vivo studies we next adapt the Vk*MYC myeloma model using a syngeneic transplant approach to facilitate the testing of novel therapeutics. In this second MYC-driven system of B-cell neoplasia, NVP-BEZ235 has only modest cytostatic activity. However, the unexpected efficacy of a commonly used drug delivery vehicle, n-methyl-2-pyrrolidone (NMP), is identified in this immunocompetent model. This NMP activity is subsequently defined as immunomodulatory with striking similarities to anti-myeloma thalidomide analogues. Moreover, a complex ‘on-target’ interaction between NVP-BEZ235 and its vehicle of administration (NMP) highlights the potential for an antagonistic relationship between mTOR inhibitors and immunomodulatory thalidomide analogues in myeloma. The divergent efficacy of NVP-BEZ235 in two closely related haematological cancer models reaffirms the importance of molecular characterisation of specific diseases when considering the clinical translation of novel kinase inhibitors. The data presented herein indicates that dual PI3K (or DDR-kinase)/mTOR inhibitor-based strategies may have efficacy in human Burkitt lymphoma, including poor risk, p53 mutated disease. Conversely, NVP-BEZ235 has a poor activity in Vk*MYC myeloma, and has the potential to attenuate immune-mediated repression of myeloma in the clinic. Careful stratification to select haematological cancers that show functional reliance on PI3K/mTOR signalling is required to effectively progress agents like NVP-BEZ235 into human patients with lymphoid neoplasia

FISH Detection of PML-RARA Fusion in ins(15;17) Acute Promyelocytic Leukaemia Depends on Probe Size

The diagnosis of acute promyelocytic leukaemia (APL) is usually confirmed by cytogenetics showing the characteristic t(15;17), but a minority of patients have a masked PML/RARA fusion. We report ten patients with APL and no evidence of the t(15;17), in whom the insertion of RARA into PML could not be demonstrated by initial FISH studies using a standard dual fusion probe but was readily identified using smaller probes. Given the need for rapid diagnosis of APL, it is important to be aware of the false negative rate for large PML/RARA FISH probes in the setting of masked rearrangements

Changes in immune cell populations following KappaMab, lenalidomide and low‐dose dexamethasone treatment in multiple myeloma

Abstract Objectives Lenalidomide (LEN) is used to treat multiple myeloma (MM) and shows in vitro synergy with KappaMab (KM), a chimeric antibody specific for Kappa Myeloma antigen, an antigen exclusively expressed on the surface of kappa‐restricted MM cells. Lenalidomide, dexamethasone (DEX) and KM control MM via multiple immunomodulatory mechanisms; however, there are several additional effects of the drug combination on immune cells. Lenalidomide can increase T cell and NKT cell cytotoxicity and dendritic cell (DC) activation in vitro. We investigated the immune cell populations in bone marrow of patients treated with KM, LEN and low‐dose DEX in kappa‐restricted relapsed/refractory MM ex vivo and assessed association of those changes with patient outcome. Methods A cohort (n = 40) of patients with kappa‐restricted relapsed/refractory MM, treated with KM, LEN and low‐dose DEX, was analysed using a mass cytometry panel that allowed identification of immune cell subsets. Clustering analyses were used to determine significant changes in immune cell populations at time periods after treatment. Results We found changes in five DC and 17 T‐cell populations throughout treatment. We showed an increase in activated conventional DC populations, a decrease in immature/precursor DC populations, a decrease in activated CD4 T cells and an increase in effector‐memory CD4 T cells and effector CD8 T cells, indicating an activated immune response. Conclusion These data characterise the effects of LEN, DEX, and KM treatment on non‐target immune cells in MM. Treatment may support destruction of MM cells by both direct action and indirect mechanisms via immune cells

An intact immune system is required for the anticancer activities of histone deacetylase inhibitors

Cell-intrinsic effects such as induction of apoptosis and/or inhibition of cell proliferation have been proposed as the major antitumor responses to histone deacetylase inhibitors (HDACi). These compounds can also mediate immune-modulatory effects that may contribute to their anticancer effects. However, HDACi can also induce anti-inflammatory, and potentially immunosuppressive, outcomes. We therefore sought to clarify the role of the immune system in mediating the efficacy of HDACi in a physiologic setting, using preclinical, syngeneic murine models of hematologic malignancies and solid tumors. We showed an intact immune system was required for the robust anticancer effects of the HDACi vorinostat and panobinostat against a colon adenocarcinoma and two aggressive models of leukemia/lymphoma. Importantly, although HDACi-treated immunocompromised mice bearing established lymphoma succumbed to disease significantly earlier than tumor bearing, HDACi-treated wild-type (WT) mice, treatment with the conventional chemotherapeutic etoposide equivalently enhanced the survival of both strains. IFN-gamma and tumor cell signaling through IFN-gamma R were particularly important for the anticancer effects of HDACi, and vorinostat and IFN-gamma acted in concert to enhance the immunogenicity of tumor cells. Furthermore, we show that a combination of vorinostat with alpha-galactosylceramide (alpha-GalCer), an IFN-gamma-inducing agent, was significantly more potent against established lymphoma than vorinostat treatment alone. Intriguingly, B cells, but not natural killer cells or CD8(+) T cells, were implicated as effectors of the vorinostat antitumor immune response. Together, our data suggest HDACi are immunostimulatory during cancer treatment and that combinatorial therapeutic regimes with immunotherapies should be considered in the clinic. (C)2013 AACR

Venous thromboembolism in primary central nervous system lymphoma during frontline chemoimmunotherapy

Background: In primary central nervous system lymphoma (PCNSL), venous thromboembolism (VTE) can cause significant morbidity and hinder chemotherapy delivery. Objectives: To assess VTE incidence, timing and adequacy of inpatient and outpatient VTE prophylaxis in patients with PCNSL receiving chemoimmunotherapy with curative intent. Patients/Methods: We reviewed patients diagnosed with PCNSL between 1997 and 2018 who received methotrexate, procarbazine, and vincristine ± Rituximab. Patient demographics, VTE prophylaxis and incidence, adverse events of anticoagulation, and survival outcomes were collected. Results: Fifty-one PCNSL patients were included (median 67 years [range, 32-87], 30 males [59%]). Thirteen patients (25%, 95% confidence interval [CI], 14-40) developed VTE at a median of 1.6 months from diagnosis (range, 0-4). Patients with Khorana Risk Score ≥2 were more likely to have VTE than those with a KRS < 2 (60% vs 15%; P = .01). Eighty-five percent had deviations from inpatient VTE prophylaxis guidelines, and outpatient prophylaxis was not routinely administered. Three patients required inferior vena cava filters. Hemorrhagic complications of anticoagulation included an intracranial hemorrhage from therapeutic anticoagulation and three cases of major bleeding from prophylactic anticoagulation. No patients died from VTE or its treatment. Conclusions: Patients with newly diagnosed PCNSL are at high risk of VTE. Further research is required into optimal VTE prophylaxis in PCNSL