MYC-driven Lymphoma Research Articles

Phosphorylation Control of p53 DNA-Binding Cooperativity Balances Tumorigenesis and Aging.

Posttranslational modifications are essential for regulating the transcription factor p53, which binds DNA in a highly cooperative manner to control expression of a plethora of tumor-suppressive programs. Here we show at the biochemical, cellular, and organismal level that the cooperative nature of DNA binding is reduced by phosphorylation of highly conserved serine residues (human S183/S185, mouse S180) in the DNA-binding domain. To explore the role of this inhibitory phosphorylation in vivo, new phosphorylation-deficient p53-S180A knock-in mice were generated. Chromatin immunoprecipitation sequencing and RNA sequencing studies of S180A knock-in cells demonstrated enhanced DNA binding and increased target gene expression. In vivo, this translated into a tissue-specific vulnerability of the bone marrow that caused depletion of hematopoietic stem cells and impaired proper regeneration of hematopoiesis after DNA damage. Median lifespan was significantly reduced by 20% from 709 days in wild type to only 568 days in S180A littermates. Importantly, lifespan was reduced by a loss of general fitness and increased susceptibility to age-related diseases, not by increased cancer incidence as often seen in other p53-mutant mouse models. For example, S180A knock-in mice showed markedly reduced spontaneous tumorigenesis and increased resistance to Myc-driven lymphoma and Eml4-Alk-driven lung cancer. Preventing phosphorylation of S183/S185 in human cells boosted p53 activity and allowed tumor cells to be killed more efficiently. Together, our data identify p53 DNA-binding domain phosphorylation as a druggable mechanism that balances tumorigenesis and aging. SIGNIFICANCE: These findings demonstrate that p53 tumor suppressor activity is reduced by DNA-binding domain phosphorylation to prevent aging and identify this phosphorylation as a potential target for cancer therapy.See related commentary by Horikawa, p. 5164.

MiR-378a-3p Is Critical for Burkitt Lymphoma Cell Growth

Simple SummaryMicroRNAs (miRNAs) are small RNAs that regulate expression of specific target genes. We observed elevated levels of miR-378a-3p in Burkitt lymphoma (BL) and studied its role in the pathogenesis of BL. Inhibition of miR-378a-3p reduced growth of BL cells, confirming its significance in BL. Identification of BL specific target genes of miR-378a-3p revealed four candidates. For two of them, MNT and IRAK4, miR-378a-dependent regulation was confirmed at the protein level. Overexpression of MNT and IRAK4 in BL cell lines resulted in a similar effect as observed upon miR-378a-3p inhibition, suggesting their involvement in the growth regulatory role of miR-378a-3p.MicroRNAs (miRNAs) are small RNA molecules with important gene regulatory roles in normal and pathophysiological cellular processes. Burkitt lymphoma (BL) is an MYC-driven lymphoma of germinal center B (GC-B) cell origin. To gain further knowledge on the role of miRNAs in the pathogenesis of BL, we performed small RNA sequencing in BL cell lines and normal GC-B cells. This revealed 26 miRNAs with significantly different expression levels. For five miRNAs, the differential expression pattern was confirmed in primary BL tissues compared to GC-B cells. MiR-378a-3p was upregulated in BL, and its inhibition reduced the growth of multiple BL cell lines. RNA immunoprecipitation of Argonaute 2 followed by microarray analysis (Ago2-RIP-Chip) upon inhibition and ectopic overexpression of miR-378a-3p revealed 63 and 20 putative miR-378a-3p targets, respectively. Effective targeting by miR-378a-3p was confirmed by luciferase reporter assays for MAX Network Transcriptional Repressor (MNT), Forkhead Box P1 (FOXP1), Interleukin 1 Receptor Associated Kinase 4 (IRAK4), and lncRNA Just Proximal To XIST (JPX), and by Western blot for IRAK4 and MNT. Overexpression of IRAK4 and MNT phenocopied the effect of miR-378a-3p inhibition. In summary, we identified miR-378a-3p as a miRNA with an oncogenic role in BL and identified IRAK4 and MNT as miR-378a-3p target genes that are involved in its growth regulatory role.

Combination of CS2164 and Venetoclax Shows Synergistic Antitumor Effect in High-Grade B-Cell Lymphomas with ConcomitantMYCandBCL2Rearrangements

High-grade B-cell lymphoma with concurrentMYCandBCL2rearrangements (HGBCL-DHL) is a rare and aggressive B-cell disorder with a high likelihood of nonresponsiveness to the front-line immunochemotherapy. Patients with HGBCL-DHL who develop a recurrent or progressive disease have limited effective therapeutics and show very poor clinical outcomes. Therefore, it calls for the development of novel targeted therapies for this specific patient populations. In this study, we showed that combination of BCL2 inhibitor venetoclax and CS2164, a novel orally active multitarget inhibitor, is a potent therapeutic strategy against HGBCL-DHL in the preclinical setting. BCL2rearrangement, a hallmark feature of HGBCL-DHL, often results in increased BCL2 protein that counteracts the proapoptotic proteins of BCL2 family, and thus blocks cell death. BCL2 blockage with venetoclax shows promising clinical responses in various human malignancies. However, resistance to venetoclax takes place following its continuous administration, suggesting that venetoclax should combine with other agents to prevent disease progression. CS2164 is originally developed by China and designed to perturb three key characteristics of neoplasms: tumor angiogenesis, cell mitosis and chronic inflammation. Our previous study demonstrated that CS2164 exerted potently antilymphoma activities in MYC-driven lymphomas, providing evidence that CS2164 could potentiate the effect of venetoclax for MYC/BCL2 driven HGBCL-DHL. In the present work, we first observed that both venetoclax and CS2164 as single agent reduced the capability of cell proliferation in HGBCL-DHL cell lines. Next, we found that CS2164 synergized with venetoclax to suppress cell proliferation and trigger cell apoptosis in HGBCL-DHLin vitro. More importantly, when compared with each single drug groups, coadministration of venetoclax and CS2164 resulted in superior suppression of HGBCL-DHL cell growth and remarkably abrogated tumor burden in a HGBCL-DHL-xenografted mouse model. The synergistic lethality of venetoclax and CS2164 towards HGBCL-DHL cells was associated with the modulation of multiple molecular mechanisms. The underlying mechanisms for the synergy of the two drugs included the blockade of Rad51 recombinase-dependent DNA repair, the perturbation of the delicate balance of BCL2 family proteins that induced mitochondrial membrane depolarization and subsequently led to the proapoptotic effect, as well as the inhibition of PI3K/AKT/mTOR pathway and MYC expression. In summary, these findings suggest that the regimen of CS2164 and venetoclax combination is highly effective to eliminate HGBCL-DHL cellsin vitroandin vivoand thus provide a rational treatment paradigm to strip HGBCL-DHL of its protection fromMYCandBCL2rearrangements. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: CS2164 originally developed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.

XPO1 Relieves MYC-Induced Replication Stress Limiting the Immunogenicity of DLBCL Cells

The soluble nuclear pore component XPO1 is a therapeutic target in DLBCL and its selective inhibitor selinexor received FDA approval in relapsed/refractory DLBCL. XPO1 inhibitors block the nuclear export of a subset of cargo proteins. Since the spectrum of XPO1 cargos varies among cell types, the mechanism underlying the anti-lymphoma effect of selinexor has not been fully elucidated. Lack of this knowledge hampers the clinical use of XPO1 inhibitors in terms of patient selection and development of rational therapeutic combinations. We reported that XPO1 is amplified and overexpressed in DLBCL. At the molecular level, we described that XPO1 overexpression functionally remodels the nuclear pore to sustain the expression of DNA damage repair proteins increasing lymphoma cells' tolerance to genotoxic stress. Here, we investigated whether this XPO1 role is relevant for the anti-lymphoma effect of selinexor. Aberrant MYC expression induces DNA damage resulting from replication stress; thus, we speculated that selinexor kills MYC expressing (MYC+) DLBCL cells by limiting their ability to tolerate replication stress. Using B-cells expressing MYC in an inducible manner, we demonstrated that exposure to selinexor (6 h) caused G2-M cell cycle arrest upon MYC expression (p<0.0001, vs. vehicle-treated cells). Cell cycle arrest was associated with increased levels of DNA damage in S-phase as measured by yH2AX (p<0.0001). Furthermore, although MYC increased the expression of key replication stress repair proteins (i.e. RAD51, WEE and BRCA1), this effect was blunted by selinexor. To determine the clinical relevance of this mechanism, we interrogated publicly available datasets of DLBCLs and found that XPO1 amplification and overexpression was associated with MYC translocation (p<0.005), double-hit lymphoma signature (p<0.005), and elevated levels of chromosomal instability (p<0.005). Overall, these observations suggest that MYC-driven lymphomas sustaining high levels of genomic instability may critically depend on XPO1. To therapeutically capitalized this mechanism, we evaluated the effect of selinexor on MYC+ DLBCLs. Clinically achievable doses of selinexor induced cell cycle arrest and apoptosis in 83% (5/6) of cell lines investigated in vitro. In a patient-derived xenograft (PDX) MYC+ DLBCL model, a human-equivalent dose of selinexor impaired tumor growth (p< 0.05, vs. vehicle) without significant toxicities. Reduced tumor growth was secondary to apoptosis as determined by TUNEL (p<0.05). To characterize the biological effects of XPO1 inhibition, we compared the transcriptome of PDX from the previous experiment. Pathway analysis of differentially expressed genes revealed that selinexor induced pathways regulating innate immune signaling. The upregulation of key genes from this pathway (i.e. DDX58, DDX60, IRF7 and STAT1) was validated by qRT-PCR. Since unrepaired DNA damage elicits an innate immune response, we hypothesized that these effects could be mechanistically linked. To test this notion, we exposed MYC+ DLBCL cells to sub-lethal doses of the DNA damaging agent etoposide, selinexor, or their combination. Expression of innate immune genes was significantly higher in cells exposed to the combination compared to either drug alone (p<0.001, for both comparisons), indicating that selinexor increases the immunogenicity of MYC+ DLBCL cells and this effect is enhanced by concomitant replication stress. Furthermore, by analyzing primary DLBCLs we found that poorly immunogenic lymphomas, characterized by an immune deserted microenvironment, expressed higher levels of XPO1 and MYC compared to DLBCL with an immune rich microenvironment (P<0.001, for both genes). To determine a potential effect of selinexor in the function of immune effector cells, we performed single cells secretomic analysis (Isolight) of circulating CD8+ T cells obtained from two DLBCL patients before and after selinexor treatment and found that selinexor increases the secretory activity of CD8+ T cells in both patients. Overall, our study reveals a key oncogenic role for XPO1 in enabling DLBCL cells to tolerate MYC-induced replication stress while limiting their immunogenicity. This suggest that patients with MYC+ DLBCL and elevated genomic instability may benefit from therapeutic approaches based on XPO1 inhibitors in combination with immunomodulatory drugs, an approach we will test prospectively. Disclosures Rutherford: AstraZeneca: Consultancy; Celgene: Consultancy; Dova: Consultancy; Genentech/Roche: Research Funding; Heron: Consultancy; Juno: Consultancy; Karyopharm: Consultancy, Research Funding; Kite: Consultancy; LAM Therapeutics: Research Funding; Regeneron: Research Funding; Seattle Genetics: Consultancy. Martin:Regeneron: Consultancy; Cellectar: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy, Research Funding; Teneobio: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Beigene: Consultancy; Sandoz: Consultancy; I-MAB: Consultancy; Morphosys: Consultancy; Kite: Consultancy; Incyte: Consultancy. Leonard:Miltenyi: Consultancy; Sutro: Consultancy; Roche/Genentech: Consultancy; BMS/Celgene: Consultancy; Regeneron: Consultancy; ADC Therapeutics: Consultancy; MEI Pharma: Consultancy; Bayer: Consultancy; Gilead/Kite: Consultancy; Karyopharm: Consultancy; GenMab: Consultancy; Epizyme: Consultancy; AstraZeneca: Consultancy. Cerchietti:Cellgene: Research Funding; Bristol Myers Squibb: Research Funding.

Abstract IA31: Modeling and targeting double-hit lymphoma

Abstract High-grade B-cell lymphoma with concurrent activation of MYC and BCL2, also known as double-hit lymphoma (DHL), shows dismal prognosis with current front-line therapies (e.g., R-CHOP), calling for the development of new therapeutic regimens. The synergy between MYC and BCL2 in lymphomagenesis is explained by the ability of BCL2 to block the proapoptotic activity of MYC, while leaving intact its proliferative potential; on this basis, we reasoned that compounds that exacerbate MYC-induced apoptosis might cooperate with BCL2 inhibitors in killing DHL cells. We previously observed that genes encoding components of the mitochondrial ribosome were coordinately activated in MYC-driven lymphoma (1) and were critical for tumor maintenance in transgenic mice (2). In line with the genetic data, inhibition of mitochondrial translation with the antibiotic tigecycline was synthetic-lethal with MYC activation and extended lifespan in lymphoma-bearing mice (2). Combining tigecycline with the BCL2 inhibitor venetoclax revealed synergy in the induction of apoptosis in human DHL cell lines, as well as marked antitumoral activity in xenografted mice (3). These preclinical data warrant the repurposing of venetoclax and tigecycline for the treatment of refractory and/or relapsed DHL. By blocking the synthesis of mitochondrially encoded proteins, tigecycline impairs the assembly of the electron-transport chain (ETC) complexes and oxidative phosphorylation. We thus hypothesized that direct ETC inhibitors might also preferentially kill MYC-overexpressing cells and synergize with venetoclax against DHL. We will present data that verify these predictions for IACS-010759, a small-molecule inhibitor of mitochondrial complex I. A current limitation for further preclinical development and mechanistic analysis lies in the lack of a bona fide mouse model of DHL. While the cooperation between MYC and BCL2 in lymphomagenesis was amply documented over almost three decades, the models described so far led to tumor onset at early stages of B-cell ontogeny; as a consequence, no model is available that reliably reproduces the development of DHL from germinal center (GC) B-cells, as observed in the clinic. In order to overcome this limitation, we combined CRE-activated alleles of MYC and BCL2 with Cgamma1-CRE, a CRE-expressing transgene that is specifically expressed in GC B cells. Concerted activation of both oncogenes led to increased expansion of GC B cells upon immunization, followed within a few months by the onset of aggressive, invasive B-cell lymphomas. Our progress in characterizing this disease model will be presented at the meeting.

BET inhibitors synergize with venetoclax to induce apoptosis in MYC-driven lymphomas with high BCL-2 expression

AbstractAlthough the MYC oncogenic network represents an attractive therapeutic target for lymphoma, MYC inhibitors have been difficult to develop. Alternatively, inhibitors of epigenetic/ transcriptional regulators, particularly the bromodomain and extraterminal (BET) family, have been used to modulate MYC. However, current benzodiazepine-derivative BET inhibitors (BETi) elicit disappointing responses and dose-limiting toxicity in relapsed/refractory lymphoma, potentially because of enrichment of high-risk molecular features and chemical backbone-associated toxicities. Consequently, novel nonbenzodiazepine BETi and improved mechanistic understanding are required. Here we characterize the responses of aggressive MYC-driven lymphomas to 2 nonbenzodiazepine BETi: PLX51107 and PLX2853. Both invoked BIM-dependent apoptosis and in vivo therapy, associated with miR-17∼92 repression, in murine Eµ-myc lymphomas, with PLX2853 exhibiting enhanced potency. Accordingly, exogenous BCL-2 expression abrogated these effects. Because high BCL-2 expression is common in diffuse large B-cell lymphoma (DLBCL), BETi were ineffective in driving apoptosis and in vivo therapy of DLBCL cell lines, mirroring clinical results. However, BETi-mediated BIM upregulation and miR-17∼92 repression remained intact. Consequently, coadministration of BETi and ABT199/venetoclax restored cell death and in vivo therapy. Collectively, these data identify BIM-dependent apoptosis as a critical mechanism of action for this class of BETi that, via coadministration of BH3 mimetics, can deliver effective tumor control in DLBCL.

MYC functions as a switch for natural killer cell-mediated immune surveillance of lymphoid malignancies

The MYC oncogene drives T- and B- lymphoid malignancies, including Burkitt’s lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). Here, we demonstrate a systemic reduction in natural killer (NK) cell numbers in SRα-tTA/Tet-O-MYCON mice bearing MYC-driven T-lymphomas. Residual mNK cells in spleens of MYCON T-lymphoma-bearing mice exhibit perturbations in the terminal NK effector differentiation pathway. Lymphoma-intrinsic MYC arrests NK maturation by transcriptionally repressing STAT1/2 and secretion of Type I Interferons (IFNs). Treating T-lymphoma-bearing mice with Type I IFN improves survival by rescuing NK cell maturation. Adoptive transfer of mature NK cells is sufficient to delay both T-lymphoma growth and recurrence post MYC inactivation. In MYC-driven BL patients, low expression of both STAT1 and STAT2 correlates significantly with the absence of activated NK cells and predicts unfavorable clinical outcomes. Our studies thus provide a rationale for developing NK cell-based therapies to effectively treat MYC-driven lymphomas in the future.

Lymphoma Angiogenesis Is Orchestrated by Noncanonical Signaling Pathways.

Tumor-induced remodeling of the microenvironment relies on the formation of blood vessels, which go beyond the regulation of metabolism, shaping a maladapted survival niche for tumor cells. In high-grade B-cell lymphoma, angiogenesis correlates with poor prognosis, but attempts to target established proangiogenic pathways within the vascular niche have been inefficient. Here, we analyzed Myc-driven B-cell lymphoma-induced angiogenesis in mice. A few lymphoma cells were sufficient to activate the angiogenic switch in lymph nodes. A unique morphology of dense microvessels emerged without obvious tip cell guidance and reliance on blood endothelial cell (BEC) proliferation. The transcriptional response of BECs was inflammation independent. Conventional HIF1α or Notch signaling routes prevalent in solid tumors were not activated. Instead, a nonconventional hypersprouting morphology was orchestrated by lymphoma-provided VEGFC and lymphotoxin (LT). Interference with VEGF receptor-3 and LTβ receptor signaling pathways abrogated lymphoma angiogenesis, thus revealing targets to block lymphomagenesis. SIGNIFICANCE: In lymphoma, transcriptomes and morphogenic patterns of the vasculature are distinct from processes in inflammation and solid tumors. Instead, LTβR and VEGFR3 signaling gain leading roles and are targets for lymphomagenesis blockade.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/80/6/1316/F1.large.jpg.

IBTK Haploinsufficiency Affects the Tumor Microenvironment of Myc-Driven Lymphoma in E-myc Mice

The tumor microenvironment is a dynamic and interactive supporting network of various components, including blood vessels, cytokines, chemokines, and immune cells, which sustain the tumor cell’s survival and growth. Murine models of lymphoma are useful to study tumor biology, the microenvironment, and mechanisms of response to therapy. Lymphomas are heterogeneous hematologic malignancies, and the complex microenvironment from which they arise and their multifaceted genetic basis represents a challenge for the generation and use of an appropriate murine model. So, it is important to choose the correct methodology. Recently, we supported the first evidence on the pro-oncogenic action of IBTK in Myc-driven B cell lymphomagenesis in mice, inhibiting apoptosis in the pre-cancerous stage. We used the transgenic Eμ-myc mouse model of non-Hodgkin’s lymphoma and Ibtk hemizygous mice to evaluate the tumor development of Myc-driven lymphoma. Here, we report that the allelic loss of Ibtk alters the immunophenotype of Myc-driven B cell lymphomas, increasing the rate of pre-B cells and affecting the tumor microenvironment in Eμ-myc mice. In particular, we observed enhanced tumor angiogenesis, increasing pro-angiogenic and lymphangiogenic factors, such as VEGF, MMP-9, CCL2, and VEGFD, and a significant recruitment of tumor-associated macrophages in lymphomas of Ibtk+/- Eμ-myc compared to Ibtk+/+ Eμ-myc mice. In summary, these results indicate that IBTK haploinsufficiency promotes Myc tumor development by modifying the tumor microenvironment.

Integrative genomic analysis focused on cell cycle genes for MYC-driven aggressive mature B-cell lymphoma.

MYC is a transcriptional factor that regulates growth and proliferation through cell cycle pathways. MYC alterations, in particular MYC rearrangements, are important in assessing the prognosis of aggressive B-cell lymphoma. In this study, we focused on the impact of nine major cell cycle genes for MYC-driven aggressive mature B-cell lymphoma and analyzed the mutational status using targeted next generation sequencing. Our 40 cases of aggressive mature B-cell lymphomas included 5 Burkitt lymphomas, 17 high-grade B-cell lymphomas and 18 diffuse large B-cell lymphomas with MYC breaks in 100%, 88% and 11%, respectively. Our data allowed a molecular classification into four categories partially independent from the histopathological diagnosis but correlating with the Ki-67 labelling index: (I) harboring TP53 and CDKN2A mutations, being highly proliferative, (II) with MYC rearrangement associated with MYC and/or ID3 mutations, being highly proliferative, (III) with MYC rearrangement combined with additional molecular changes, being highly proliferative, and (IV) with a diverse pattern of molecular alterations, being less proliferative. Taken together, we found that mutations of TP53, CDKN2A, MYC and ID3 are associated with highly proliferative B-cell lymphomas that could profit from novel therapeutic strategies.

MCL-1 Inhibition By the Selective MCL-1 Inhibitor AMG-176 Induces in Vitro Activity Against Burkitt Lymphoma Cell Lines and Synergistically Enhances the Cytotoxic Effect of Chemotherapy and BH3 Mimetics

Background: Although >90% of Burkitt Lymphoma (BL) patients will be cured by intensive multi-agent immunochemotherapy, relapsed and refractory BL patients have poor prognoses demonstrating the need to identify novel therapies. Myeloid cell leukemia-1 (MCL-1) is a pro-survival protein within the BCL-2 family that has been investigated as a therapeutic target in several leukemia and lymphoma pre-clinical models and clinical studies. Analysis of pediatric BL tumors has implicated MCL-1 in BL pathogenesis (Giulino-Roth et al., Blood 2012) and targeting MCL-1 may be effective in MYC-driven lymphomas (Kelly et al., Genes Dev 2014). High MCL-1 expression has also been associated with therapy resistance and can confer resistance to BH3 mimetic agents (Carrington et al., Cell Death Differ 2017). AMG176, a selective MCL-1 inhibitor, is an effective promoter of apoptosis and is currently under investigation in clinical trials for relapsed and refractory multiple myeloma, lymphoma, and acute myeloid leukemia. Hypothesis: We hypothesized that MCL-1 inhibition with AMG176 will promote apoptosis and increase sensitivity of BL therapy-sensitive (TSCL) and therapy-resistant (TRCL) cell lines to other chemotherapy drugs. Methods: A panel of TSCLs (Raji, Ramos and Daudi) and one TRCL (Raji 4RH) BL cell lines were exposed to increasing concentrations of AMG176 as a single agent and in combination with Doxorubicin or Venetoclax at different time points. Cell viability was analyzed using PrestoBlue. Synergy in drug combination exposures was determined by Calcusyn software and reported as a combination index (CI) value with values increasingly <1 indicating increasing synergistic activity. Apoptosis induction was analyzed by flow cytometry for Annexin V (AV) and Sytox-7AAD and by western blotting for PARP. Effects of MCL-1 inhibition on the expression of other pro- and anti-apoptotic BCL-2 family member proteins was assessed by western blotting. AMG176 mechanism(s) of action was evaluated by co-immunoprecipitation followed by western blotting for pro-apoptotic BCL-2 family members known to be bound by MCL-1. Results: Single agent AMG176 exposure of BL cell lines exhibited a dose and time-dependent decrease in viable cells. 48hr IC50 values of Ramos (0.3097uM) and Raji (1.652uM) showed an increased sensitivity to AMG176 as compared to Daudi (9.616uM) and Raji 4RH (12.45uM). Exposure of cells to AMG176 also resulted in altered expression levels of pro-survival and pro-apoptotic proteins of the BCL-2 family proteins. Co-immunoprecipitation performed after 48hrs of AMG176 exposure exhibited a decrease in MCL-1:BIM and MCL-1:BAK complexes in Raji and Ramos cells promoting a pro-apoptotic environment. After 48hr exposure to AMG176, induction of apoptosis was observed in a dose-dependent manner in all cell lines as evidenced by increased PARP cleavage on western blot and AV-Sytox positivity on flow cytometry. To investigate the ability of MCL-1 inhibition to enhance the activity of cytotoxic chemotherapy, BL cells were exposed to AMG176 in combination with Doxorubicin. Daudi and Raji exhibited strong synergy with peak synergistic activity observed at 72hrs (Daudi CI values <0.1 at all AGM176 concentrations with 0.1-0.2uM Doxorubicin; Raji CI ranged from 0.233-0.295 at multiple AMG176 concentrations with <0.4uM Doxorubicin) while a minimal effect was observed in Ramos and Raji 4RH. As high MCL-1 is associated with resistance to Venetoclax by sequestering free BIM following Venetoclax exposure, the combination of AMG176 and Venetoclax was investigated and also showed significant synergy in BL cells. A minimal effect was observed in Daudi and Raji 4RH in Venetoclax combination but Ramos and Raji, the more AMG176 sensitive cell lines, showed peak synergy at 24hrs at high doses of Venetoclax (Ramos CI values ranged from 0.3-0.478; Raji CI ranged from 0.511 to 0.67). Conclusion: MCL-1 inhibition with AMG176 demonstrates dose- and time-dependent anti-lymphoma activity impairing in vitro proliferation and inducing apoptosis in BL cells. AMG176 exhibits synergistic activity in combination with cytotoxic chemotherapy in both therapy-sensitive and therapy-resistant BL cell lines and synergistically enhances response to the BH3 mimetic agent Venetoclax. These findings highlight the potential of MCL-1 inhibition as a therapeutic option in BL. This work was supported by T35 NIH training grant (T35AI089693). Disclosures No relevant conflicts of interest to declare.

The whole-genome landscape of Burkitt lymphoma subtypes

AbstractBurkitt lymphoma (BL) is an aggressive, MYC-driven lymphoma comprising 3 distinct clinical subtypes: sporadic BLs that occur worldwide, endemic BLs that occur predominantly in sub-Saharan Africa, and immunodeficiency-associated BLs that occur primarily in the setting of HIV. In this study, we comprehensively delineated the genomic basis of BL through whole-genome sequencing (WGS) of 101 tumors representing all 3 subtypes of BL to identify 72 driver genes. These data were additionally informed by CRISPR screens in BL cell lines to functionally annotate the role of oncogenic drivers. Nearly every driver gene was found to have both coding and non-coding mutations, highlighting the importance of WGS for identifying driver events. Our data implicate coding and non-coding mutations in IGLL5, BACH2, SIN3A, and DNMT1. Epstein-Barr virus (EBV) infection was associated with higher mutation load, with type 1 EBV showing a higher mutational burden than type 2 EBV. Although sporadic and immunodeficiency-associated BLs had similar genetic profiles, endemic BLs manifested more frequent mutations in BCL7A and BCL6 and fewer genetic alterations in DNMT1, SNTB2, and CTCF. Silencing mutations in ID3 were a common feature of all 3 subtypes of BL. In vitro, mass spectrometry–based proteomics demonstrated that the ID3 protein binds primarily to TCF3 and TCF4. In vivo knockout of ID3 potentiated the effects of MYC, leading to rapid tumorigenesis and tumor phenotypes consistent with those observed in the human disease.

1011 - THE ROLE OF MUTANT P53 IN TUMOR DEVELOPMENT AND THE RESPONSE OF MALIGNANT CELLS TO ANTI-CANCER THERAPEUTICS

p53 is a highly potent tumour suppressor that acts through activation of several biological processes, including apoptotic cell death, cell growth arrest and cell senescence. In accordance with its critical role in tumour suppression, the p53 gene is the most frequently mutated gene in human cancers. Mutant p53 cancers are usually relatively resistant to chemotherapeutic drugs and such patients therefore often have a poor prognosis. Despite several decades of research into the functions of wild-type (wt) p53 in normal cells and mutant p53 in tumour cells, we still do not have a clear understanding of how mutations in p53 contribute to the development and sustained growth of cancers. There is evidence to support the notion that mutant p53 acts only to blocks wt p53 tumour suppressor functions (dominant negative effect; DNE) but there are also reports that mutant p53 has de novogain-of-function properties that additionally promote tumour growth. Of critical importance is the question of whether mutant p53 contributes to the sustained growth of established tumours, thereby validating whether abrogating its expression would be a promising therapeutic approach. Clarification of this point has profound implications for the future development and clinical success of cancer drugs that aim to directly target and degrade mutant p53. We have investigated the role of mutant p53 in aggressive MYC-driven lymphomas, focusing on how it functions during tumour development and also the response to conventional and targeted cancer therapeutics.

An early Myc-dependent transcriptional program orchestrates cell growth during B-cell activation.

Upon activation, lymphocytes exit quiescence and undergo substantial increases in cell size, accompanied by activation of energy-producing and anabolic pathways, widespread chromatin decompaction, and elevated transcriptional activity. These changes depend upon prior induction of the Myc transcription factor, but how Myc controls them remains unclear. We addressed this issue by profiling the response to LPS stimulation in wild-type and c-myc-deleted primary mouse B-cells. Myc is rapidly induced, becomes detectable on virtually all active promoters and enhancers, but has no direct impact on global transcriptional activity. Instead, Myc contributes to the swift up- and down-regulation of several hundred genes, including many known regulators of the aforementioned cellular processes. Myc-activated promoters are enriched for E-box consensus motifs, bind Myc at the highest levels, and show enhanced RNA Polymerase II recruitment, the opposite being true at down-regulated loci. Remarkably, the Myc-dependent signature identified in activated B-cells is also enriched in Myc-driven B-cell lymphomas: hence, besides modulation of new cancer-specific programs, the oncogenic action of Myc may largely rely on sustained deregulation of its normal physiological targets.

Targeting translation initiation by synthetic rocaglates for treating MYC-driven lymphomas.

MYC-driven lymphomas, especially those with concurrent MYC and BCL2 dysregulation, are currently a challenge in clinical practice due to rapid disease progression, resistance to standard chemotherapy and high risk of refractory disease. MYC plays a central role by coordinating hyperactive protein synthesis with upregulated transcription in order to support rapid proliferation of tumor cells. Translation initiation inhibitor rocaglates have been identified as the most potent drugs in MYC-driven lymphomas as they efficiently inhibit MYC expression and tumor cell viability. We found that this class of compounds can overcome eIF4A abundance by stabilizing target mRNA-eIF4A interaction that directly prevents translation. Proteome-wide quantification demonstrated selective repression of multiple critical oncoproteins in addition to MYC in B cell lymphoma including NEK2, MCL1, AURKA, PLK1, and several transcription factors that are generally considered undruggable. Finally, (−)-SDS-1–021, the most promising synthetic rocaglate, was confirmed to be highly potent as a single agent, and displayed significant synergy with the BCL2 inhibitor ABT199 in inhibiting tumor growth and survival in primary lymphoma cells in vitro and in patient-derived xenograft mouse models. Overall, our findings support the strategy of using rocaglates to target oncoprotein synthesis in MYC-driven lymphomas.

IBTK contributes to B-cell lymphomagenesis in E\u03bc-myc transgenic mice conferring resistance to apoptosis

Increasing evidence supports the involvement of IBTK in cell survival and tumor growth. Previously, we have shown that IBTK RNA interference affects the wide genome expression and RNA splicing in cell-type specific manner. Further, the expression of IBTK gene progressively increases from indolent to aggressive stage of chronic lymphocytic leukemia and decreases in disease remission after therapy. However, the role of IBTK in tumorigenesis has not been elucidated. Here, we report that loss of the murine Ibtk gene raises survival and delays tumor onset in Eμ-myc transgenic mice, a preclinical model of Myc-driven lymphoma. In particular, we found that the number of pre-cancerous B cells of bone marrow and spleen is reduced in Ibtk−/−Eμ-myc mice owing to impaired viability and increased apoptosis, as measured by Annexin V binding, Caspase 3/7 cleavage assays and cell cycle profile analysis. Instead, the proliferation rate of pre-cancerous B cells is unaffected by the loss of Ibtk. We observed a direct correlation between Ibtk and myc expression and demonstrated a Myc-dependent regulation of Ibtk expression in murine B cells, human hematopoietic and nonhematopoietic cell lines by analysis of ChIP-seq data. By tet-repressible Myc system, we confirmed a Myc-dependent expression of IBTK in human B cells. Further, we showed that Ibtk loss affected the main apoptotic pathways dependent on Myc overexpression in pre-cancerous Eμ-myc mice, in particular, MCL-1 and p53. Of note, we found that loss of IBTK impaired cell cycle and increased apoptosis also in a human epithelial cell line, HeLa cells, in Myc-independent manner. Taken together, these results suggest that Ibtk sustains the oncogenic activity of Myc by inhibiting apoptosis of murine pre-cancerous B cells, as a cell-specific mechanism. Our findings could be relevant for the development of IBTK inhibitors sensitizing tumor cells to apoptosis.

Transient stabilization, rather than inhibition, of MYC amplifies extrinsic apoptosis and therapeutic responses in refractory B-cell lymphoma

Therapeutic targeting of initiating oncogenes is the mainstay of precision medicine. Considerable efforts have been expended toward silencing MYC, which drives many human cancers including Burkitt lymphomas (BL). Yet, the effects of MYC silencing on standard-of-care therapies are poorly understood. Here we found that inhibition of MYC transcription renders B-lymphoblastoid cells refractory to chemotherapeutic agents. This suggested that in the context of chemotherapy, stabilization of Myc protein could be more beneficial than its inactivation. We tested this hypothesis by pharmacologically inhibiting glycogen synthase kinase 3β (GSK-3β), which normally targets Myc for proteasomal degradation. We discovered that chemorefractory BL cell lines responded better to doxorubicin and other anti-cancer drugs when Myc was transiently stabilized. In vivo, GSK3 inhibitors (GSK3i) enhanced doxorubicin-induced apoptosis in BL patient-derived xenografts (BL-PDX), as well as in murine MYC-driven lymphoma allografts. This enhancement was accompanied by and required deregulation of several key genes acting in the extrinsic, death-receptor-mediated apoptotic pathway. Consistent with this mechanism of action, GSK3i also facilitated lymphoma cell killing by a death ligand TRAIL and by a death receptor agonist mapatumumab. Thus, GSK3i synergizes with both standard chemotherapeutics and direct engagers of death receptors and could improve outcomes in patients with refractory lymphomas.

B cell primary immune responses.

B cell primary immune responses.

The B-cell receptor in control of tumor B-cell fitness: Biology and clinical relevance.

Surface expression of a functional B cell antigen receptor (BCR) is essential for the survival and proliferation of mature B cells. Most types of B-cell lymphoproliferative disorders retain surface BCR expression, including B-cell non-Hodgkin lymphomas (B-NHL) and chronic lymphocytic leukemia (CLL). Targeting BCR effectors in B-NHL cell lines in vitro has indicated that this signaling axis is crucial for malignant B cell growth. This has led to the development of inhibitors of BCR signaling, which are currently used for the treatment of CLL and several B-NHL subtypes. Recent studies based on conditional BCR inactivation in a MYC-driven mouse B-cell lymphoma model have revisited the role of the BCR in MYC-expressing tumor B cells. Indeed, lymphoma cells losing BCR expression continue to grow unless subjected to competition with their BCR-expressing counterparts, which causes their elimination. Here, we discuss the molecular nature of the fitness signal delivered by the BCR to MYC-expressing malignant B cells, ensuring their preferential persistence within a rapidly expanding tumor population. We also review growing evidence of Ig-negative cases belonging to several B-NHL subtypes and CLL, and discuss the clinical implications of these findings in relation to an emerging picture of clinical resistances to anti-BCR therapies.

Changes in long-range rDNA-genomic interactions associate with altered RNA polymerase II gene programs during malignant transformation

The three-dimensional organization of the genome contributes to its maintenance and regulation. While chromosomal regions associate with nucleolar ribosomal RNA genes (rDNA), the biological significance of rDNA-genome interactions and whether they are dynamically regulated during disease remain unclear. rDNA chromatin exists in multiple inactive and active states and their transition is regulated by the RNA polymerase I transcription factor UBTF. Here, using a MYC-driven lymphoma model, we demonstrate that during malignant progression the rDNA chromatin converts to the open state, which is required for tumor cell survival. Moreover, this rDNA transition co-occurs with a reorganization of rDNA-genome contacts which correlate with gene expression changes at associated loci, impacting gene ontologies including B-cell differentiation, cell growth and metabolism. We propose that UBTF-mediated conversion to open rDNA chromatin during malignant transformation contributes to the regulation of specific gene pathways that regulate growth and differentiation through reformed long-range physical interactions with the rDNA.