Presence Of MYC Research Articles

HYbrid DegRAding Copolymer (HYDRAC): Heterobifunctional polymers for targeted degradation of undruggable proteins.

22 Background: Of the ~20,000 proteins encoded by the human genome, only a small subset can be modulated via traditional pharmacological approaches. Included in the list of “undruggable” proteins are ones with pivotal roles in cancer. We aim to expand the arsenal of compounds able to affect the activity of these therapeutically relevant targets by introducing a modular proteomimetic platform technology designed to carry side chains consisting of targeting warheads and recruiters of cellular protein degradation machinery. We term these compounds HYbrid DegRAding Copolymers (HYDRACs) in reference to the multiplexing of different protein binders and degradation inducing sequences. Peptides arranged as HYDRACs exhibit favorable emergent properties including resistance to proteolysis, high levels of cell uptake, and payload-specific activity. Initial studies were conducted focusing on a undruggable transcription factor MYC, which is widely dysregulated in human cancers. Methods: A library of HYDRACs and control polymers were synthesized and were cell-penetrating with antiproliferative effects at sub-micromolar IC50s in a formulation- and MYC-dependent manner across multiple cell lines. Results: HYDRAC treatment resulted in significant decreases in MYC protein levels, which was rescued by proteasome inhibitor MG132 and neddylation inhibitor MLN4924, without changes in mRNA levels. RNAseq showed selective overexpression of MYC pathway genes only in HYDRAC treated cells, confirming on-target effects. Circular dichroism showed strong HYDRAC-MYC interactions, which was supported by the presence of MYC following pull-down of biotin-terminated HYDRACs. Removal of the RRRG degron, scrambling the H1 sequence, and disassociation of the two domains on separate polymers all abrogated MYC-degradation. Mice bearing transplanted Myc-CaP tumors showed delayed tumor growth and reduced tumor cell proliferation following HYDRAC treatment. Mice bearing luminescent Luc-MV-411 tumors treated with Cy5.5-labeled HDRACs showed selective tumor accumulation with detectable fluorescent signal in the tumor up to 72 hours following a single IP administration. The versatility of the platform was demonstrated by substituting the degron for recruiters of three different E3 ligases (VHL, KEAP1, and CRBN), which all maintained ability to degrade MYC. HYDRACs were also able to selectively degrade other protein targets including alpha-syn and RAS. Conclusions: We present a platform technology that addresses challenges inherent to small molecule drug design. HYDRACs have the potential to dramatically alter the drug discovery landscape, allowing for development of modulators of "undruggable" targets. We envision the HYDRAC platform as a generalizable approach to designing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for target protein degradation.

Double-hit and double-expressor primary central nervous system lymphoma: Experience from North India of an infrequent but aggressive variant.

High-grade non-Hodgkin B-cell lymphoma is an aggressive mature B-cell lymphoma that depicts poor treatment response and worse prognosis. The presence of MYC and B-cell lymphoma 2 (BCL2) and/or B-cell lymphoma 6 (BCL6) rearrangements qualifies for triple-hit and double-hit lymphomas (THL/DHL), respectively. We attempted to explore the incidence, distribution, and clinical characteristics of the primary high-grade B-cell lymphoma of the central nervous system (CNS) in our cohort from North India. All the histologically confirmed cases of primary CNS diffuse large B-cell lymphoma (PCNS-DLBCL) over a period of 8 years were included. Cases showing MYC and BCL2 and/or BCL6 expression on immunohistochemistry (IHC) (double- or triple-expressor) were further analyzed by fluorescence in situ hybridization for MYC, BCL2 and /or BCL6 rearrangements. The results were correlated with other clinical and pathological parameters, and outcome. Of total 117 cases of PCNS-DLBCL, there were seven (5.9%) cases of double/triple-expressor lymphomas (DEL/TEL) (six double- and one triple-expressor) with median age of 51 years (age range: 31-77 years) and slight female predilection. All were located supratentorially and were of non-geminal center B-cell phenotype. Only triple-expressor case (MYC+/BCL2+/BCL6+) demonstrated concurrent rearrangements for MYC and BCL6 genes indicating DHL (n = 1, 0.85%), while none of the double-expressors (n = 6) showed MYC, BCL2, or BCL6 rearrangements. The mean overall survival of the DEL/TEL was 48.2 days. DEL/TEL and DHL are uncommon in CNS; mostly located supratentorially and are associated with poor outcome. MYC, BCL2, and BCL6 IHC can be used as an effective screening strategy for ruling out double/ triple-expressor PCNS-DLBCLs.

MYC and TP53 Alterations but Not MAPK Pathway Mutations Are Common Oncogenic Mechanisms in Follicular Dendritic Cell Sarcomas.

Despite their stromal origin, follicular dendritic cells (FDCs) share many functions with hematopoietic system cells. FDC neoplasms are currently classified by the World Health Organization along with those of a histiocytic nature. However, the molecular alterations driving oncogenesis in FDC sarcomas (FDCSs) are beginning to be unveiled and do not seem to concur with those described in histiocytic neoplasms, namely MAPK pathway activation. To identify molecular alterations driving tumorigenesis in FDCS. We investigated the role of MYC and TP53 in FDC-derived tumor oncogenesis and assessed comprehensively the status of the MAPK pathway in 16 FDCSs, 6 inflammatory pseudotumor (IPT)-like FDCSs, and 8 IPTs. MYC structural alterations (both amplifications and rearrangements) were identified in 5 of 14 FDCSs (35.7%), all associated with MYC overexpression. TP53 mutations were identified in 4 of 14 FDCSs (28.6%), all of which displayed intense and diffuse p53 expression. None of these alterations were identified in any IPT-like FDCSs or in IPT cases. No MAPK pathway gene alterations were identified in any of the cases studied. The presence of MYC and TP53 alterations and the lack of association with Epstein-Barr virus segregate classical FDCS from IPT-like FDCS, pointing at different oncogenic mechanisms in both entities. Our results suggest a possible oncogenic role of MYC and TP53 alterations in FDCS. The absence of MAPK pathway alterations confirms the lack of a significant role of this pathway in the oncogenesis of FDC-derived neoplasms.

HOW I TREAT DOUBLE-HIT LYMPHMOMA AND HGBL, NOS

The new world health organization (WHO) classification on lymphoid neoplasms, the WHO-HAEM5, renames the former group that double-hit lymphomas were in as “diffuse large B-cell lymphoma/high-grade B-cell lymphoma with MYC and BCL2 rearrangements (DLBCL/HGBL-MYC/BCL2)”. This is mainly to highlight that the presence of MYC and BCL2 rearrangements form a unique phenotype, different than the MYC and BCL6 rearrangements (present in the former classification). Those lymphomas are composed of large or intermediate or blastoid cells, with aggressive clinical course and tendency to be resistant to standard chemotherapy. It's a group ideal for new therapies, such as the bispecifics and CAR T-cells, but lack data to support this since are underrepresented in clinical trials. Retrospective studies, with its inherit bias, consistently points to worst prognosis and poor outcomes with standard RCHOP treatment. How to best approach this hard-to-treat lymphoma is still a matter of debate. Roughly 65% of patients with DLBCL are cure with 6 cycles of RCHOP. When considering this regimen for HGBL, event-free survival (EFS) has been reported as low as 20% in 3 years. More intensive regimens, like R-DA-EPOCH and R-CODOX/M-IVAC, could increase this response, based on retrospective studies, with EFS 3y close to 80%. The role of autologous transplant as consolidation is controversial, and it's not routinely indicated. However, there are data that patients treated with RCHOP could increase progression-free survival (PFS) with this strategy, perhaps eliminating the difference between more intensive regimens. The lack of a direct comparison in a randomize phase 3 study between RCHOP or more intensive protocols precludes a firm conclusion. In the Alliance/CALGB 50303 study, that compared RCHOP with R-DAEPOCH in patients with DLBCL and PMBCL, there were no differences in 2y PFS between arms. But the number of patients with MYC rearrangement was too small to any conclusion regarding HGBL. Dunleavy et al conducted a phase 2 study with R-DA-EPOCH in 53 patients with MYC-rearranged DLBCL (24 were double-hit). EFS 4y was 71% and overall-survival (OS) 4y was 77%. Although this looks pretty good compared to the historic RCHOP, it's not a randomize study. New therapies have emerged as possible rescue in the relapsed/refractory DLBCL population, a group of patients with a dismal prognosis. The chimeric antigen receptor (CAR) T-cells have become a new standard of care for those patients, when available. Albeit with a small number of patients, the three main products (axi-cell, tisa-cell and liso-cell), used for rescue of DLBCL patients, had shown activity against HGBL. That holds true in latter lines and as a first salvage treatment, as the recent trials comparing with autologous transplant. The zuma-12 is a phase 2 study with axi-cell as first-line of treatment with high-risk DLBCL patients, a population enriched with HGBL. Early reports are impressive, with nearly 80% of complete remissions. However, long term follow-up will be necessary to see with the responses are durable. Bispecifics are other very important players on that field, with the first reports of high activity in high-risk DLBCL, even after CAR T-cell failure. HGBL is an aggressive form of lymphoma, with tendency of a worst prognosis with conventional treatment. Intensive regimens seem to fare better than RCHOP, although with more toxicity and no randomize studies supporting this indication. New treatments, mainly CAR T-cells and bispecifics, are very promising and possibly will became standard of care for such patients but were in the therapy algorithm is still to be decide.

Abstract 2502: Unravelling the heterogenous molecular landscape of pediatric post-transplant lymphoproliferative disorders

Abstract The genetic landscape of post-transplant lymphoproliferative disorders (PTLD) in pediatric population has not been fully elucidated. This absence of information raises the question whether therapeutic strategies should be the same as for their counterparts in immunocompetent (IC) patients. The aim of this study was to characterize genetically and immunophenotypically pediatric monomorphic PTLD. Thirty-nine monomorphic PTLD ≤19 years-old (mean 10y, gender 25 male/14 female) were recruited and analyzed for germinal center markers, IRF4 and EBER expression. Presence of MYC, PAX5, IRF4, BCL2, BCL6 and 11q alterations was investigated by FISH. Additional molecular studies included clonality, copy number (CN) arrays, cell of origin-COO (Nanostring) and mutational analyses (Custom 167 lymphoma related genes panel, SureSelectXT, Agilent). Twenty-nine patients received solid organ transplantation and eight were hematopoietic stem cell transplant recipients. The mean time from transplant to PTLD diagnosis was 34 months (range 2-170) and the estimated 5-year overall survival (5y-OS) rate was 67%. Patients that received a solid organ had a better prognosis than hematopoietic stem cell transplant recipients (5y-OS 83% vs. 38%, p=0.03). Thirty-three cases were classified as diffuse large B-cell lymphoma (DLBCL) and six as Burkitt lymphoma (BL). Thirty-two cases had extranodal localization, 21 of which in the gastrointestinal tract. Among the DLBCL, 24/28 cases had an ABC/non-GC COO phenotype and the six BL were GCB. EBER was positive in 33/37 cases. Five out of six BL and one DLBCL had MYC rearrangements, while no 11q alterations or other rearrangements were observed. Ten out of the 23 pediatric monomorphic PTLD studied displayed CN alterations (mean 1.6 alt/case; range 0-12). Comparative analyses showed that pediatric PTLD had lower genetic complexity than BL (Scholtysik, 2010) and DLBCL (Ramis-Zaldivar, 2020) in IC patients and adult-PTLD (Ferreiro, 2016; Rinaldi, 2010) and lacked characteristic CN alterations of those groups. Regarding the mutational profile, all 6 PTLD-BL carried MYC mutations in addition to ID3 (4 cases), ARID1A (2 cases) or CCND3 (1 case) and a higher mutational burden than PTLD-DLBCL (12.3 vs 6.2, p=0.01). PTLD-DLBCL showed a very heterogeneous profile characterized by a lower number of mutations than their counterparts in IC patients (2.4 vs 6.5, p=0.01). Pathway enrichment analysis revealed that epigenetic modifiers and NOTCH pathway (4 cases each) were the most recurrently affected. Two out of 20 cases were classified as N1 according to LymphGen (Wright, 2020) algorithm while the rest remained undetermined. The mutational profile of pediatric PTLD-BL is similar to that observed in IC patients whereas PTLD-DLBCL are less complex than their counterpart in IC children and present a very heterogeneous mutational landscape with enrichment in NOTCH pathway mutations. Citation Format: Julia Salmeron, Natalia Castrejón-de-Anta, Pilar Guerra-Garcia, Joan Enric Ramis-Zaldivar, Mónica López-Guerra, Dolors Colomer, Francisco Diaz-Crespo, Marta Garrido, Javier Menarguez, Maria del Mar Andrés, Eugenia Garcia-Fernandez, Margarita Llavador, Noelia Garcia, Blanca Gonzalez-Farré, Idoia Martin-Guerrero, Carmen Garrido, Itziar Astigarraga, Alba Fernández, Jaime Verdú-Amorós, Soledad González-Muñíz, Berta Gonzalez, Verónica Celis, Elias Campo, Olga Balagué, Itziar Salaverria. Unravelling the heterogenous molecular landscape of pediatric post-transplant lymphoproliferative disorders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2502.

Incremental Value of FDG PET/CT in Aggressive High Grade B Cell lymphoma with TdT Expression

High-grade B-cell lymphoma, an aggressive form of Non-Hodgkin's Lymphoma, is known as a double or triple hit lymphoma based on the presence of MYC and BCL2 without or with BCL6 genetic rearrangements, respectively. It carries a poorer prognosis, compared to other variants of B-cell lymphoma, and its management also differs which requires more intensive chemotherapy in contrast to the routine regimen. Terminal deoxynucleotidyl transferase (TdT), a marker of immaturity is commonly expressed in B-cell lymphoblastic leukemia or lymphoma (B cell ALL) which is absent in mature forms of B-cell lymphoma. The TdT is expressed in high-grade B-cell lymphoma; therefore, it poses a classification and management dilemma, which should be accurately differentiated from B-cell ALL and mandates molecular analysis. Herein, we report a case of a 52-year-old female with biopsy reported as high-grade B-cell lymphoma with TdT expression. She was referred for Fluor-deoxyglucose (FDG) Positron Emission Tomography-Computed Tomography (PET/CT) scan for staging in the absence of molecular analysis for B-cell ALL. It was diagnosed as lymphoma on FDG PET/CT based on its characteristic findings of extensive extranodal involvement of multiple organs with no significant lymphadenopathy establishing the incremental value of FDG PET/CT scan, which helped the clinician to arrive at a conclusion.

A Versatile In Vivo System to Study Myc in Cell Reprogramming.

Cellular reprogramming is a process by which adult differentiated cells lose their identity and are converted into pluripotent stem cells, known as induced pluripotent stem (iPS) cells. This process can be achieved in vitro and in vivo and is relevant for many fields including regenerative medicine and cancer. Cellular reprogramming is commonly induced by the ectopic expression of a transcription factor cocktail composed by Oct4, Sox2, Klf4, and Myc (abbreviated as OSKM), and its efficiency and kinetics are strongly dependent on the presence of Myc. Here, we describe a versatile method to study reprogramming in vivo based on the use of adeno-associated viral (AAV) vectors, which allows the targeting of specific organs and cell types. This method can be used to test Myc mutations or genes that may replace Myc, or be combined with different Myc regulators. In vivo reprogramming can be scored by the presence of teratomas and the isolation of in vivoiPS, thereby providing a simple surrogate for the function of Myc in dedifferentiation and stemness. Our protocol can be divided into five steps: (1) intravenous inoculation of AAV vectors; (2) monitoring the animals until the appearance of teratomas; (3) analysis of teratomas; (4) histopathological analysis of mouse organs; and (5) isolation of in vivo-generated iPS cells from teratomas, blood, and bone marrow. The information obtained by this in vivo testing platform may provide relevant information on the role of Myc in tissue regeneration, stemness, and cancer.

Double-Hit Signature with TP53 Abnormalities Predicts Poor Survival in Patients with Germinal Center Type Diffuse Large B-Cell Lymphoma Treated with R-CHOP

Purpose: We performed detailed genomic analysis on 87 cases of de novo diffuse large B-cell lymphoma of germinal center type (GCB DLBCL) to identify characteristics that are associated with survival in those treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Experimental Design: The cases were extensively characterized by combining the results of immunohistochemistry, cell-of-origin gene expression profiling (Nanostring), double-hit gene expression profiling (DLBCL90), fluorescence in situ hybridization (FISH) cytogenetic analysis for double/triple-hit lymphoma, copy number analysis (CNA), and targeted deep sequencing using a custom mutation panel of 334 genes. Results: We identified four distinct biologic subgroups with different survivals, and with similarities to the genomic classifications from two large retrospective studies of DLBCL. Patients with the double-hit signature but no abnormalities of TP53, and those lacking EZH2 mutation and/or BCL2 translocation, had an excellent prognosis. However, patients with an EZB-like profile had an intermediate prognosis, whereas those with TP53 inactivation combined with the double-hit signature had an extremely poor prognosis. This latter finding was validated using two independent cohorts. Conclusions: We propose a practical schema to utilize genomic variables to risk-stratify patients with GCB DLBCL. This schema provides a promising new approach to identify high-risk patients for new and innovative therapies.

RNA Regulator of Lipogenesis (RROL) Is a Novel Lncrna Mediating Protein-Protein Interaction at Gene Regulatory Loci Driving Lipogenic Programs in Multiple Myeloma

Long noncoding RNA (lncRNAs) are key epigenetic factors that drive the origin and progression of human cancers via mechanisms that are largely unknown. We have previuosly reported the clinical significance of a lncRNA signature in multiple myeloma (MM) as independent risk predictor for clinical outcome; and recently identified a lncRNA RROL (RNA Regulator of Lipogenesis) with impact on MM cell proliferation. Here we describe a unique regulatory function for RROL in the control of gene networks involved in the de novo lipogenesis (DNL) pathway, ultimately impacting MM cell growth and survival. Based on growth and survival impact of RROL depletion, we performed integrated transcriptomic analysis of RNA-seq data after RROL depletion in MM cell lines and CD138+ patient MM cells, and identified a set of significantly modulated metabolic genes including the acetyl Co-A Carboxylase 1 (ACC1) gene, encoding a rate-limiting enzyme of the DNL pathway. This metabolic pathway converts nutrients into fatty acids serving for energy storage or biosynthesis of membranes and signaling molecules. Consistent with the transcriptional control of ACC1, we have observed that RROL inhibition in cell lines and primary MM cells significantly decreased the incorporation of C14-radiolabeled glucose into de novo synthesized lipids. Importantly, supplementation with exogenous palmitate, the main downstream product of DNL pathway, rescued the growth inhibitory effect of RROL depletion on MM cells, further confirming the importance of the DNL pathway in the oncogenic activity of RROL in MM. To understand the molecular mechanism through which RROL regulates ACC1 expression and its metabolic axis, we evaluated the RROL interactome in MM cells. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to the identification of MYC as relevant direct partner of RROL. These results were further validated by qRT-PCR analysis of MYC-bound RNA obtained through RNA immunoprecipitation (RIP) assay. Using experimental model of conditional MYC KD (P493-6), we found that RROL exerts regulatory activity on ACC1 only in the presence of MYC. Mapping of MYC genomic occupancy by ChIP-seq and gene expression after MYC KD in MM cells revealed that ACC1 is a direct transcriptional target of MYC in cells expressing RROL. These data indicate that RROLand MYC cooperate in the transcriptional control of ACC1. Moreover, we found that RROL itself is transcriptionally regulated by MYC, suggesting the existence of a feed-forward regulatory loop in which MYC enhances the expression of RROL that, in turn, drives MYC transcriptional activity to ACC1. We hypothesized that RROL may shape the protein interacting network of MYC to confer specificity for ACC1 promoter. To this end, we performed mass spectrometry analysis of MYC interactome in three MM cell lines in the presence or in the absence of RROL and identified the transcriptional modulator WDR82 as RROL-dependent MYC partner. Interestingly, WDR82 directly interacts with RROL as assessed in the RPPD and RNA Y3H assays, and transcriptionally regulates ACC1 in RROL-dependent manner. These data indicate that RROL catalyzes the interaction of MYC with the transcriptional modulator WDR82 to form a transcriptional ternary complex regulating ACC1 expression. To therapeutically antagonize the RROL lipogenic signaling we have pre-clinically tested small molecule inhibitors of ACC1 (ACC1i). We have observed significant anti-MM activity of ACC1i in vitro in a large panel of MM cell lines and primary MM cells from patients; and in vivo in mouse models of human MM including the localized subcutaneous model and the disseminated model that establish a more aggressive systemic disease. Importantly, we have now developed clinically applicable ASOs and small molecule-like compounds to directly target RROL in MM cells. These studies are ongoing and will be presented. In conclusion, we here report a unique regulatory function of a novel lncRNA supporting MM cell growth via its control of the lipogenic metabolic axis. The availability of oral inhibitors of ACC1 as well as the ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM. Disclosures Fulciniti: NIH: Research Funding. Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

A Clinico-Genotypic Prognostic Index for De Novo Composite Diffuse Large B-Cell Lymphoma Arising from Follicular Lymphoma in Asian patients treated in the Rituximab Era

Composite follicular lymphoma with diffuse large B-cell lymphoma (FL/DLBCL) is uncommonly found on lymph node biopsy and represents a rare haematological malignancy. We aim to examine clinico-pathological features of patients with FL/DLBCL and investigate predictors of survival outcome. We included in our retrospective study patients with histologically-proven FL/DLBCL at diagnosis (n = 106) and who were subsequently treated with rituximab-based chemoimmunotherapy from 2002–2017 at the National Cancer Centre. The cohort consisted of 34 women and 72 men with a median age of 59 years (range, 24–82). In a multivariate model inclusive of known clinico-pathological parameters at diagnosis, advanced stage (p = 0.0136), presence of MYC and/or BCL6 rearrangement (p = 0.0376) and presence of B symptoms (p = 0.0405) were independently prognostic for worse overall survival (OS). The only remaining independent prognostic variables for worse OS after including first-line treatment data in the model were use of chemotherapy regimens other than R-CHOP (p = 0.0360) and lack of complete response to chemotherapy (p < 0.0001) besides the presence of B symptoms (p = 0.0022). We generated a Clinico-Genotypic Index by point-wise addition of all five adverse parameters (score of 0–1, 2, 3, 4–5) which revealed four prognostic risk groups with a predicted 5-year OS of 100%, 62%, 40% and 0% (p < 0.0001) accounting for 50.0%, 24.5%, 18.9% and 6.6% of the cohort respectively. We propose that R-CHOP should be the recommended first-line regimen for composite FL/DLBCL.

Drug therapy for double-hit lymphoma.

Double-hit lymphoma (DHL) is a rare type of aggressive B-cell lymphoma defined as a high-grade B-cell lymphoma (HGBCL) with the presence of MYC, BCL2 and/or BCL6 rearrangements. Patients usually present with rapidly progressive and advanced stage of disease and, commonly, with extranodal involvement. Typically, patients become refractory to standard R-CHOP, and more aggressive regimens such as DA-EPOCH-R, R-hyperCVAD or CODOX-R regimens are typically needed. MYC is considered an “undruggable” mutation. Recent evidence suggests that pathogenic mechanisms associated with MYC could be potential targets. In this review, we also discuss the role of hematopoietic stem cell transplantation (HCT) and chimeric antigen receptor (CAR) T-cell therapy in DHL. We also discuss the role of potential novel agents such as BCL2 inhibitors, checkpoint inhibitors, bromodomain and extraterminal (BET) family inhibitors, Pi3K inhibitors, and others.

ZUMA-12: A phase 2 multicenter study of axicabtagene ciloleucel (axi-cel) as a first-line therapy in patients (pts) with high-risk large B-cell lymphoma (LBCL).

TPS7574 Background: Pts with LBCL who have persistent disease assessed by dynamic PET after rituximab-based induction therapy have an increased risk of death (Casasnovas, et al. Blood. 2017). Axi-cel is an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy approved for the treatment of pts with relapsed/refractory LBCL with ≥ 2 prior systemic therapies. In ZUMA-1, the registrational study of axi-cel in pts with refractory LBCL, the objective response rate (ORR) was 91% (70% complete response [CR] rate) in pts with double-expressor or high-grade LBCL with ongoing responses in 48% after a median follow-up of 27.1 mos (Locke FL, et al. Lancet Oncol 2019). Furthermore, pts with fewer prior lines of therapy and lower tumor burden had higher rates of ongoing responses and manageable safety (Locke et al. ASCO 2018. 3039). ZUMA-12 will investigate the efficacy and safety of axi-cel as a first-line therapy in newly diagnosed pts with high-risk LBCL who have PET-positive disease after 2 cycles of induction therapy. Methods: This Phase 2 study has a planned enrollment of ≈40 pts aged ≥ 18 y with high-risk LBCL, defined by the presence of MYC and BCL2 and/or BCL6 translocations by FISH or an IPI score ≥ 3 any time before enrollment, and an ECOG performance status of 0 – 1. Before enrollment, pts must have a Deauville score of 4 – 5 (Barrington SF et al. J Clin Oncol. 2014) after 2 cycles of chemoimmunotherapy that includes an anti-CD20 monoclonal antibody and anthracycline. After leukapheresis, pts with bulky or rapidly progressing disease may receive optional non-chemotherapy bridging therapy. Following conditioning therapy with cyclophosphamide (500 mg/m2) and fludarabine (30 mg/m2) for 3 days, pts will receive a single infusion of axi-cel at a target dose of 2 × 106 CAR T cells/kg. The primary endpoint is investigator-assessed CR rate per the Lugano classification (Cheson et al. J Clin Oncol. 2014). Key secondary endpoints include ORR, duration of response, event-free survival, progression-free survival, overall response, safety, relapse with CNS disease and levels of blood CAR T cells and serum cytokines over time. Accrual is ongoing. Clinical trial information: NCT03761056.

Keratin 23 Is a Peroxisome Proliferator-Activated Receptor Alpha-Dependent, MYC-Amplified Oncogene That Promotes Hepatocyte Proliferation.

Chronic activation of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARA) promotes MYC-linked hepatocellular carcinoma (HCC) in mice. Recent studies have shown that MYC can function as an amplifier of transcription where MYC does not act as an "on-off" switch for gene expression but rather accelerates transcription rates at active promoters by stimulating transcript elongation. Considering the possibility that MYC may amplify the expression of PPARA target genes to potentiate cell proliferation and liver cancer, gene expression was analyzed from livers of wild-type and liver-specific Myc knockout (MycΔHep ) mice treated with the PPARA agonist pirinixic acid. A subset of PPARA target genes was amplified in the presence of MYC, including keratin 23 (Krt23). The induction of Krt23 was significantly attenuated in MycΔHep mice and completely abolished in Ppara-null mice. Reporter gene assays and chromatin immunoprecipitation confirmed direct binding of both PPARA and MYC to sites within the Krt23 promoter. Forced expression of KRT23 in primary hepatocytes induced cell cycle-related genes. These data indicate that PPARA activation elevates MYC expression, which in turn potentiates the expression of select PPARA target genes involved in cell proliferation. Finally, KRT23 protein is highly elevated in human HCCs. Conclusion: These results revealed that MYC-mediated transcriptional potentiation of select PPARA target genes, such as Krt23, may remove rate-limiting constraints on hepatocyte growth and proliferation leading to liver cancer.

Médulloblastome. Anatomie pathologique

Medulloblastomas, embryonal neuroepithelial tumors developed in the cerebellum or brain stem, are mainly observed in childhood. The treatment of WHO-Grade IV tumors depends on stratifications that are usually based on postoperative data, histopathological subtype, tumor extension and presence of MYC or NMYC amplifications. Recently, molecular biology studies, based on new technologies (i.e. sequencing, transcriptomic, methylomic) have introduced genetic subtypes integrated into the latest WHO-2016 neuropathological classification. According to this classification, the three genetic groups WNT, SHH, with or without mutated TP53 gene, and non-WNT/non-SHH, comprising subgroups 3 and 4, are recalled in this review. The contribution of immunohistochemistry to define these groups is specified. The four histopathological groups are detailed in comparison to the WHO-2007 classification and the molecular data: classic medulloblastoma, desmoplastic/nodular medulloblastoma, medulloblastoma with extensive nodularity, and large cell/anaplastic medulloblastoma. The groups defined on genetic and histopathological grounds are not strictly concordant. Depending on the age of the patients, their correlations are different, as well as their role in the management and prognosis of these tumors. Other embryonal tumors, for which new classifications are in progress and gliomas may be confused with a medulloblastoma and the elements of the differential diagnosis of these entities are discussed. This evolution in classification fully justifies ongoing structuring procedures such as histopathological review (RENOCLIP) and the organization of molecular biology platforms.

Direct HPV E6/Myc interactions induce histone modifications, Pol II phosphorylation, and hTERT promoter activation.

Human Papillomavirus Viruses (HPVs) are associated with the majority of human cervical and anal cancers and 10-30% of head and neck squamous carcinomas. E6 oncoprotein from high risk HPVs interacts with the p53 tumor suppressor protein to facilitate its degradation and increases telomerase activity for extending the life span of host cells. We published previously that the Myc cellular transcription factor associates with the high-risk HPV E6 protein in vivo and participates in the transactivation of the hTERT promoter. In the present study, we further analyzed the role of E6 and the Myc-Max-Mad network in regulating the hTERT promoter. We confirmed that E6 and Myc interact independently and that Max can also form a complex with E6. However, the E6/Max complex is observed only in the presence of Myc, suggesting that E6 associates with Myc/Max dimers. Consistent with the hypothesis that Myc is required for E6 induction of the hTERT promoter, Myc antagonists (Mad or Mnt) significantly blocked E6-mediated transactivation of the hTERT promoter. Analysis of Myc mutants demonstrated that both the transactivation domain and HLH domain of Myc protein were required for binding E6 and for the consequent transactivation of the hTERT promoter, by either Myc or E6. We also showed that E6 increased phosphorylation of Pol II on the hTERT promoter and induced epigenetic histone modifications of the hTERT promoter. More important, knockdown of Myc expression dramatically decreased engagement of acetyl-histones and Pol II at the hTERT promoter in E6-expressing cells. Thus, E6/Myc interaction triggers the transactivation of the hTERT promoter by modulating both histone modifications, Pol II phosphorylation and promoter engagement, suggesting a novel mechanism for telomerase activation and a new target for HPV- associated human cancer.

Targeting the PI3K/mTOR Pathway in Lymphoma with PQR309 and PQR620: Single Agent Activity and Synergism with the BCL2 Inhibitor Venetoclax

Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016).Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax.Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells.Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations.The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P < 0.005) and RIVA (D14, D16, D19, D21; P < 0.005).Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P < 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P < 0.001) and PQR309 (D7, D9, D11; P < 0.005) alone.Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. DisclosuresHillmann:PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

The Prognostic Value of MYC, BCL2 and BCL6 Overexpression Evaluated By Immunohistochemistry (IHC) in De-Novo Diffuse Large B Cell Lymphoma (DLBCL) Treated with Rituximab-CHOP

Introduction: MYC, BCL2 and BCL6 overexpression, assessed by IHC, with the latter conferring a better prognosis, have been reported to be a prognostic factor in DLBCL, but data are not consistent and sometimes contradictory.The aim of the present study was to assess the prognostic impact of overexpression of MYC, BCL2, and BCL6 in a retrospective cohort of de-novo DLBCL, selected for an high proliferation index (MIB1 ≥70%), treated consecutively with R-CHOP regimen.Methods: Patients with de-novo DLBCL diagnosed between January 2010 and December 2013 were included into the study. Inclusion criteria were: high proliferation index MIB1 ≥ 70% and a full course of R-CHOP regimen. Paraffin-embedded tumor samples were collected and investigated using immunohistochemistry (IHC) for MYC, BCL2 and BCL6. Fluorescence in situ hybridization (FISH) is ongoing. MYC/BCL2+ or MYC/BCL6+ double expression cases were identified if they had rearrangements of MYC and BCL2 or BCL6. MYC immunochemistry was done on TMA sections using the antibody clone Y69. BCL2 and BCL6 staining had been evaluated previously at diagnosis. Tumor cells were defined positive for MYC and BCL2 or BCL6 protein expression by immunostaining if >40%, >40% and >25% of cells showed positive expression, respectively. Progression free survival curves (PFS) were estimated using the Kaplan-Meier method and compared between groups using the log-rank test and Cox models.Results: One hundred and sixty seven patients are evaluable for clinical characteristics and 69/167 had paraffin embedded tumor samples available for immunohistochemistry at the time of present analysis. Clinical characteristics of the 69 cases were: median age 66 years (IQR 57;73), 45 (65%) male, 47 (68%) stage III-IV, 35 (54%) with elevated LDH levels and 46 (67%) at International Prognostic Index (IPI) high intermediate or high risk. Overexpression of MYC was detected in 28 cases (41%), 50 (72%) and 38 (55%) showed BCL2 and BCL6 overexpression respectively. Nineteen (28%) cases showed MYC/BCL2+ and 17 (25%) MYC/BCL6+ double expression. With a median follow up of 26 months, the median 2-years PFS was 59%. Overexpression of MYC and BCL2 proteins and low expression of BCL6 were associated with an inferior 2-years PFS in univariate analysis: MYC- vs MYC+ 64% vs 55%; BCL2- vs BCL2+ 71% vs 56%; BCL6+ vs BCL6- 61% vs 54%. In a Cox multivariate regression model adjusted for IPI and age, MYC overexpression, BCL2 positivity and BCL6 negativity showed prognostic relevance as significant independent indicators with different risk (Hazard ratio 2.53 for MYC+, 2.08 for BCL2+ and 1.62 for BCL6-). Established that the three variable contributed with different risk in the multivariate analysis, an IHC sum additive score of 0-5 was calculated proportionally to the coefficient estimated (coefficient [Log hazard ratio] 0.92 for MYC+, 0.73 for BCL2+ and 0.48 for BCL6-), assigning an individual risk of 2 points for MYC or BCL2 positivity and 1 point for BCL6 negativity. Two years-PFS was significantly different between all separate groups (Hazard ratio for unit increase 1.57 95% CI 1.11-2.22, p=0.01). After pooling scores 0-1 (with or without BCL6), 2 (presence of MYC or BCL2 only), and 3-4-5 (MYC+/BCL6-, BCL2+/BCL6-, MYC+/BCL2+, MYC+/BCL2+/BCL6-) 2-yrs PFS rates were different across the three groups: 100% vs 64% vs 50% (log rank p= 0.04) (figure 1).Conclusion: Our data showed, with the limits of a small sample size, that MYC overexpression alone or with high expression of BCL2 and/or low expression of BCL6 correlates with a worse prognosis independently by IPI score in a cohort of DLBCL selected for high proliferation index and treated with R-CHOP. Assessment of MYC, BCL2 and BCL6 expression by IHC represents a rapid, inexpensive, and reproducible technique. These results need to be confirmed in our complete series of 167 patients (analysis ongoing) and validated prospectively in a larger cohort, using standardized staining and scoring methodologies. Thus, MYC and BCL2 represent relevant biomarkers that should be tested in future clinical trials using novel effective and targeted agents in order to improve the prognosis of DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 5044: MYC drives formation of primative neuro-ectodermal tumors in human neural stem cells derived from multiple brain regions.

Abstract Primitive neuroectodermal tumors (PNET) are thought to arise from neural stem cells. They are highly aggressive tumors, and they can arise in different regions of the brain. A subset of PNET has genomic amplification of MYC, and the MYC-regulator LIN28A is known to define a group of PNET. The AKT/PTEN pathway is commonly altered in aggressive brain tumors. hTERT is expressed in the majority of PNET. Deletion or mutation of p53 is also a common event in PNET. We hypothesized that a combination of dominant negative R248Wp53, c-myc, AKT and hTERT would transform human neural stem cells into a model for PNET. We used lentiviral vectors to infect human neural stem cells derived from hindbrain and cortex with the same packages of oncogenic elements. After antibiotic selection, we performed western blotting and qPCR to verify the presence of MYC and constitutively active AKT. Increased TP53 expression on western blot was used as a proxy for inactivation of the p53 pathway by dominant negative R248W p53. Increased hTERT expression was verified by qPCR. Hindbrain neural stem cells transduced with MYC/R248Wp53/AKT/hTERT proliferated at an increased rate compared to control cells as measured by BrdU incorporation (Hindbrain pBABE vs. Hindbrain MYC/R248Wp53/AKT/hTERT: 4% vs. 50% BrdU positive, t-test p=0.001). Similar results were observed in cortex derived human neural stem cells transduced with MYC/R248Wp53/AKT/hTERT (Cortex pBABE vs Cortex MYC/R248Wp53/AKT/hTERT: 5% vs. 24% BrdU positive, t-test p=0.027). We then tested the tumor forming ability of cortex and hindbrain derived human neural stem cells, by performing orthotopic intracranial injections on immunocompromised mice. No tumors formed from human neural stem cells infected with one or two oncogenic elements. However, both hindbrain and cortex derived human neural stem cells transformed with MYC /R248Wp53/hTERT, and MYC/R248Wp53/hTERT/AKT formed aggressive orthotopic xenograft tumors with histologic characteristics of PNET. The latency of tumor formation decreased with increasing addition of oncogenes. These PNET-like tumors were synaptophysin positive, GFAP negative, had a high proliferative index, and showed signs of leptomeningal dissemination. Taken together, these experiments show that human neural stem cells from different brain regions, when transduced with the same package of oncogenes, form PNET-like orthotopic xenograft tumors. These data suggest that MYC's transforming power may trump loco-regional differences in neural stem and progenitor cells. Citation Format: Isabella Taylor, Ulf Kahlert, Jarek Maciaczyk, Guido Nikkhah, Charles Eberhart, Eric H. Raabe. MYC drives formation of primative neuro-ectodermal tumors in human neural stem cells derived from multiple brain regions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5044. doi:10.1158/1538-7445.AM2013-5044

Metabolic changes in cancer cells upon suppression of MYC

BackgroundCancer cells engage in aerobic glycolysis and glutaminolysis to fulfill their biosynthetic and energetic demands in part by activating MYC. Previous reports have characterized metabolic changes in proliferating cells upon MYC loss or gain of function. However, metabolic differences between MYC-dependent cancer cells and their isogenic differentiated counterparts have not been characterized upon MYC suppression in vitro.ResultsHere we report metabolic changes between MYC-dependent mouse osteogenic sarcomas and differentiated osteoid cells induced upon MYC suppression. While osteogenic sarcoma cells increased oxygen consumption and spare respiratory capacity upon MYC suppression, they displayed minimal changes in glucose and glutamine consumption as well as their respective contribution to the citrate pool. However, glutamine significantly induced oxygen consumption in the presence of MYC which was dependent on aminotransferases. Furthermore, inhibition of aminotransferases selectively diminished cell proliferation and survival of osteogenic sarcoma MYC-expressing cells. There were minimal changes in ROS levels and cell death sensitivity to reactive oxygen species (ROS)-inducing agents between osteoid cells and osteogenic sarcoma cells. Nevertheless, the mitochondrial-targeted antioxidant Mito-Vitamin E still diminished proliferation of MYC-dependent osteogenic sarcoma cells.ConclusionThese data highlight that aminotransferases and mitochondrial ROS might be attractive targets for cancer therapy in MYC-driven tumors.

Risk Adapted-High Dose Therapies Modulate the Impact of Biological Classification in Diffuse Large B Cell Lymphoma Prognosis. Analysis of Biological Markers in Patients From Clinical Trials in Geltamo and Gotel Spanish Collaborative Groups.

AbstractAbstract 2672 IntroductionSeveral molecular markers are now available for prognostic stratification in patients with DLBCL. Cell of Origin classification based on immunohistochemistry (IHC), microRNA expression and Fluorescent In situ Hybridization (FISH) analysis for C-MYC, and BCL2 traslocations have been demonstrated to provide relevant prognostic information in patients treated with conventional chemoiimunotherapy (R-CHOP and R-CHOP-like) regimens. However, few studies have tried to validate these results in cohorts of patients treated with high dose regimens adapted to the clinical risk. Patients, Material and MethodsHere we have performed a comprehensive biological analysis of a series of 157 patients enrolled in 4 clinical trials from GELTAMO and GOTEL Spanish Collaborative Groups. Patients were treated with first-line high dose regimens (R-CHOP 14: 76 patients; dose-adjusted EPOCH-R: 42 patients and MegaCHOP-R and bone marrow transplantation: 39 patients) according to pre-treatment clinical risk based on IPI score. Centralized review of the histopathological diagnosis and laboratory tests were performed in a single institution. All tests were determined in the FFPE diagnostic sample. We have analyzed the clinical impact on outcome (OS and PFS) of the Cell of Origin Classification based on IHC (using Hans, Choi and Visco-Young algorithms), the presence of MYC, BCL2 and BCL6 translocations by FISH and previously defined microRNA expression signatures based on RT-PCR (Montes-Moreno et al, Blood 2011). A retrospective series of 240 R-CHOP 21 treated DLBCL cases was used for comparison. ResultsOS and PFS at the median follow-up time (60 months) were 84 % (±3,4%) and 79% (±3,5%). No significant differences were found in OS and PFS according to the study protocol. Among the clinical parameters included in IPI score, only advanced age (>60 years) was predictive of poor OS and PFS in the univariant analysis (p <0.05). IPI risk stratification showed a trend for statistical significance for PFS (p 0.052). Significant differences in the distribution of GCB and NON-GC/ABC cases were found according to the classification method with percentages of GCB cases ranging from 41 to 52% and NONGC/ABC cases from 40 to 52% (χ2<0.001). However, none of the immunohistochemical algorithms was predictive for OS nor PFS in the whole series nor after protocol stratification. Interestingly ABC type DLBCL prognosis was significantly improved in this series when compared with the R-CHOP 21 treated control cohort (p <0.005), while GCB type DLBCL prognosis was not altered. By FISH, 6 cases (3, 8%), 19 (12,1%) and 27 (17,2%) of the evaluable cases had MYC, BCL2 and BCL6 translocations respectively. Only 3 cases of double hit lymphoma were found (1 with MYC snd BCL2 translocations, 2 cases with MYC and BCL6 translocations). None of these genetic alterations was predictive for OS nor PFS.Among the biological markers here analyzed, only miRNA expression signatures were found of prognostic value for OS and PFS and independent in the multivariant analysis with age (Relative Risk for miRNAs 3.41 and 2.47 for OS and PFS respectively, compared to 3.20 and 3.02 for advanced age, p < 0,05). ConclusionsThe prognostic impact of specific biological factors in DLBCL is dependent on the therapeutic regimen. Risk adapted-high dose protocols abolish the prognostic effect of the Cell Of Origin classification as determined by immunohistochemistry with recently developed algorithms. Genetic alterations in CMYC, BCL2 and BCL6 are rare and carry no prognostic information in this series of patients. Only age and microRNA expression signatures add prognostic information in patients with DLBCL treated with risk-adapted high dose regimens. Disclosures:No relevant conflicts of interest to declare.