Rapid generation of human B-cell lymphomas via combined expression of Myc and Bcl2 and their use as a preclinical model for biological therapies

Although numerous mouse models of B-cell malignancy have been developed via the enforced expression of defined oncogenic lesions, the feasibility of generating lineage-defined human B-cell malignancies using mice reconstituted with modified human hematopoietic stem cells (HSCs) remains unclear. In fact, whether human cells can be transformed as readily as murine cells by simple oncogene combinations is a subject of considerable debate. Here, we describe the development of humanized mouse model of MYC/BCL2-driven ‘double-hit’ lymphoma. By engrafting human HSCs transduced with the oncogene combination into immunodeficient mice, we generate a fatal B malignancy with complete penetrance. This humanized-MYC/BCL2-model (hMB) accurately recapitulates the histopathological and clinical aspects of steroid-, chemotherapy- and rituximab-resistant human ‘double-hit’ lymphomas that involve the MYC and BCL2 loci. Notably, this model can serve as a platform for the evaluation of antibody-based therapeutics. As a proof of principle, we used this model to show that the anti-CD52 antibody alemtuzumab effectively eliminates lymphoma cells from the spleen, liver and peripheral blood, but not from the brain. The hMB humanized mouse model underscores the synergy of MYC and BCL2 in ‘double-hit’ lymphomas in human patients. Additionally, our findings highlight the utility of humanized mouse models in interrogating therapeutic approaches, particularly human-specific monoclonal antibodies.Kathy and Curt Marble Cancer Research FundSingapore-MIT Alliance for Research and TechnologyNational Institutes of Health (U.S.) (Grant R01-CA128803)Virginia and Daniel K. Ludwig Graduate FellowshipNational Institute of General Medical Sciences (U.S.) (Medical Scientist Training Program Grant T32GM007753)MIT School of Science (Cancer Research Fellowship

Genetic Ablation of Bcl-x Attenuates Invasiveness without Affecting Apoptosis or Tumor Growth in a Mouse Model of Pancreatic Neuroendocrine Cancer

Tumor cell death is modulated by an intrinsic cell death pathway controlled by the pro- and anti-apoptotic members of the Bcl-2 family. Up-regulation of anti-apoptotic Bcl-2 family members has been shown to suppress cell death in pre-clinical models of human cancer and is implicated in human tumor progression. Previous gain-of-function studies in the RIP1-Tag2 model of pancreatic islet carcinogenesis, involving uniform or focal/temporal over-expression of Bcl-xL, demonstrated accelerated tumor formation and growth. To specifically assess the role of endogenous Bcl-x in regulating apoptosis and tumor progression in this model, we engineered a pancreatic β-cell-specific knockout of both alleles of Bcl-x using the Cre-LoxP system of homologous recombination. Surprisingly, there was no appreciable effect on tumor cell apoptosis rates or on tumor growth in the Bcl-x knockout mice. Other anti-apoptotic Bcl-2 family members were expressed but not substantively altered at the mRNA level in the Bcl-x-null tumors, suggestive of redundancy without compensatory transcriptional up-regulation. Interestingly, the incidence of invasive carcinomas was reduced, and tumor cells lacking Bcl-x were impaired in invasion in a two-chamber trans-well assay under conditions mimicking hypoxia. Thus, while the function of Bcl-x in suppressing apoptosis and thereby promoting tumor growth is evidently redundant, genetic ablation implicates Bcl-x in selectively facilitating invasion, consistent with a recent report documenting a pro-invasive capability of Bcl-xL upon exogenous over-expression

Immunoglobulin, glucocorticoid, or combination therapy for multisystem inflammatory syndrome in children: a propensity-weighted cohort study

Background Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments. Methods The Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370. Findings We enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2–11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75–1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58–1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91–1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70–1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11–0·20]; p<0·0001) and glucocorticoids alone (0·68 [0·50–0·93]; p=0·014) versus intravenous immunoglobulin alone. Persistent fever (from day 2 onward) was less common with intravenous immunoglobulin plus glucocorticoids compared with either intravenous immunoglobulin alone (OR 0·50 [95% CI 0·38–0·67]; p<0·0001) or glucocorticoids alone (0·63 [0·45–0·88]; p=0·0058). Coronary artery aneurysm occurrence and resolution did not differ significantly between treatment groups. Interpretation Recovery rates, including occurrence and resolution of coronary artery aneurysms, were similar for primary treatment with intravenous immunoglobulin when compared to glucocorticoids or intravenous immunoglobulin plus glucocorticoids. Initial treatment with glucocorticoids appears to be a safe alternative to immunoglobulin or combined therapy, and might be advantageous in view of the cost and limited availability of intravenous immunoglobulin in many countries. Funding Imperial College London, the European Union's Horizon 2020, Wellcome Trust, the Medical Research Foundation, UK National Institute for Health and Care Research, and National Institutes of Health

Critical B-lymphoid cell intrinsic role of endogenous MCL-1 in c-MYC-induced lymphomagenesis

Evasion of apoptosis is critical for tumorigenesis, and sustained survival of nascent neoplastic cells may depend upon the endogenous levels of pro-survival BCL-2 family members. Indeed, previous studies using gene-targeted mice revealed that BCL-XL, but surprisingly not BCL-2, is critical for the development of c-MYC-induced pre-B/B lymphomas. However, it remains unclear whether another pro-survival BCL-2 relative contributes to their development. MCL-1 is an intriguing candidate, because it is required for cell survival during early B-lymphocyte differentiation. It is expressed abnormally high in several types of human B-cell lymphomas and is implicated in their resistance to chemotherapy. To test the B-cell intrinsic requirement for endogenous MCL-1 in lymphoma development, we conditionally deleted Mcl-1 in B-lymphoid cells of Eμ-Myc transgenic mice. We found that MCL-1 loss in early B-lymphoid progenitors delayed MYC-driven lymphomagenesis. Moreover, the lymphomas that arose when MCL-1 levels were diminished appeared to have been selected for reduced levels of BIM and/or increased levels of BCL-XL. These results underscore the importance of MCL-1 in lymphoma development and show that alterations in the levels of other cell death regulators can compensate for deficiencies in MCL-1 expression

Individual and overlapping roles of BH3-only proteins Bim and Bad in apoptosis of lymphocytes and platelets and in suppression of thymic lymphoma development

BH3-only proteins, such as Bim and Bad, contribute to tissue homeostasis by initiating apoptosis in a cell type- and stimulus-specific manner. Loss of Bim provokes lymphocyte accumulation in vivo and renders lymphocytes more resistant to diverse apoptotic stimuli and Bad has been implicated in the apoptosis of haematopoietic cells upon cytokine deprivation. To investigate whether their biological roles in apoptosis overlap, we generated mice lacking both Bim and Bad and compared their haematopoietic phenotype with that of the single-knockout and wild-type (wt) animals. Unexpectedly, bad(-/-) mice had excess platelets due to prolonged platelet life-span. The bim(-/-)bad(-/-) mice were anatomically normal and fertile. Their haematopoietic phenotype resembled that of bim(-/-) mice but lymphocytes were slightly more elevated in their lymph nodes. Although resting B and T lymphocytes from bim(-/-)bad(-/-) and bim(-/-) animals displayed similar resistance to diverse apoptotic stimuli, mitogen activated bim(-/-)bad(-/-) B cells were more refractory to cytokine deprivation. Moreover, combined loss of Bim and Bad-enhanced survival of thymocytes after DNA damage and accelerated development of γ-irradiation-induced thymic lymphoma. Unexpectedly, their cooperation in the thymus depended upon thymocyte-stromal interaction. Collectively, these results show that Bim and Bad can cooperate in the apoptosis of thymocytes and activated B lymphocytes and in the suppression of thymic lymphoma development

Puma and to a lesser extent Noxa are suppressors of Myc-induced lymphomagenesis

Evasion of apoptosis contributes importantly to c-Myc-induced tumorigenesis. The BH3-only Bcl-2 family members Puma and Noxa are critical pro-apoptotic transcriptional targets of p53, a major mediator of Myc-induced apoptosis and suppressor of Myc-induced tumorigenesis. Hence, we have explored the impact of their individual or combined loss on myc-driven lymphomagenesis. Notably, Puma deficiency both increased B-lineage cells and accelerated the development of B lymphoma, accompanied by leukaemia, but not of pre-B lymphoma. Noxa deficiency alone also increased B-lineage cells but did not accelerate lymphomagenesis. However, its deficiency combined with loss of one puma allele produced more rapid onset of both pre-B and B lymphomas than did loss of a single puma allele alone. Nevertheless, the acceleration evoked by loss of both genes was not as marked as that caused by p53 heterozygosity. These results show that Puma imposes a significant, and Noxa a minor barrier to c-Myc-driven lymphomagenesis. They also indicate that additional BH3-only proteins probably also drive Myc-induced apoptosis and that non-apoptotic functions of p53 may contribute substantially to its tumour suppressor role