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Plasma cells can provide lifelong immunity by the continued production of high-affinity, antigen-specific antibody. Low et al. reveal that the transcription factor IRF4 acts within established plasma cells to prevent apoptosis and maintain cellular identity and mitochondrial homeostasis. The transcription factor interferon regulatory factor 4 (IRF4) is critical for the development, maintenance, and function of plasma cells. The mechanism by which IRF4 exerts its action in mature plasma cells has been elusive due to the death of all such cells upon IRF4 loss. While we identify apoptosis as a critical pathway for the death of plasma cells caused by IRF4 loss, we also determine that IRF4 did not regulate the intrinsic apoptotic pathway directly. By using an inducible IRF4 deletion system in the presence of the overexpression of anti-apoptotic BCL2, we identify genes whose expression is coordinated by IRF4 and that in turn specify plasma cell identity and mitochondrial homeostasis.Copyright © 2019 The Authors

Plasma cells can provide lifelong immunity by the continued production of high-affinity, antigen-specific antibody. Low et al. reveal that the transcription factor IRF4 acts within established plasma cells to prevent apoptosis and maintain cellular identity and mitochondrial homeostasis. The transcription factor interferon regulatory factor 4 (IRF4) is critical for the development, maintenance, and function of plasma cells. The mechanism by which IRF4 exerts its action in mature plasma cells has been elusive due to the death of all such cells upon IRF4 loss. While we identify apoptosis as a critical pathway for the death of plasma cells caused by IRF4 loss, we also determine that IRF4 did not regulate the intrinsic apoptotic pathway directly. By using an inducible IRF4 deletion system in the presence of the overexpression of anti-apoptotic BCL2, we identify genes whose expression is coordinated by IRF4 and that in turn specify plasma cell identity and mitochondrial homeostasis.Copyright © 2019 The Authors

Introduction Current DLBCL prognostic scores are poor predictors of individual risk with 26% of "very good" and "good" risk patients having unfavourable outcomes. Hypercalcaemia has been associated with poor outcomes in several cancers including multiple myeloma, but is not included in current DLBCL prognostic scoring models. Aim To determine the prognostic significance of hypercalcaemia in newly diagnosed DLBCL and identify relationships between hypercalcaemia and other established prognostic variables including cell of origin, and the components of the revised international prognostic index (R-IPI including impaired performance status, older age, advanced stage, elevated lactate dehydrogenase, and extranodal involvement. Methods Retrospective cohort study at two academic healthcare networks in Melbourne, Australia. All patients with a newly diagnosed DLBCL by WHO 2008 criteria were eligible for inclusion. Cases were identified from hospital lymphoma databases. Only cases with adequate clinical information including baseline characteristics, therapy received and outcomes were included. Cell of origin was determined by the modified Hans criteria. At Monash Health, patients were included from September 2002 until December 2015, and from Western Health, patients were included from January 2012 until December 2015. Hypercalcaemia was defined as a serum calcium >2.6mmol/L within 30 days prior to, or 15 days following the diagnosis of DLBCL. Serum calcium levels were corrected for hypoalbuminaemia if present. Immunohistochemical cell of origin was recorded using modified Hans criteria (Meyer et al. 2011). Survival correlates were estimated by Kaplan-Meier log rank method. Multivariable analysis was performed by Cox regression. Relationships between serum calcium and other covariates were examined by t test for continuous variables and chi for categorical variables. This project was approved by the human research review boards at each institution. Results A total of 514 p

The transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) have recently been found to be targeted for destruction by the immunomodulatory drugs (IMiDs). However, the role of Ikaros and Aiolos and why their loss leads to multiple myeloma (MM) cell death remains unclear. We have used CRISPRCas9 genome editing to delete IKZF1 and IKZF3 in human MM cell lines to gain further insight into their downstream gene regulatory networks. Consistent with the action of the IMiDs, loss of either of these genes resulted in both a G1/S cell cycle arrest and induction of apoptosis. This was not dependent on the subsequent reduction of the IRF4-MYC "axis", as neither IRF4 or MYC were consistently downregulated across all cell lines examined (despite cell death occurring) and overexpression of either factor failed to rescue for Ikaros loss. This led us to further investigate the transcriptional changes resulting from deletion of Ikaros, Aiolos or treatment with the IMiD lenalidomide using RNA-sequencing. Supporting their central role in the action of the IMiDs, significant overlap was seen on loss of Ikaros, Aiolos, or lenalidomide treatment; with loss of Ikaros/Aiolos accounting for approximately 75% of differential gene expression following lenalidomide. Importantly both Ikaros and Aiolos were found to repress the expression of interferon stimulated genes (ISGs) and their loss led to the activation of an interferon-like response, possibly contributing to cell death. In keeping with this, treatment with lenalidomide and low dose IFNb resulted in synergistic cell death. Furthermore, CD38 appeared to be both an ISG and repressed target of Ikaros/Aiolos through their interaction with the nucleosome remodelling and deacetylase complex, and its expression is increased at both mRNA and protein level on their loss. Given recent clinical studies have shown improved outcomes with combination treatment with lenalidomide and the anti-CD38 monoclonal antibody daratumumab, we wondered whether th

Introduction Current DLBCL prognostic scores are poor predictors of individual risk with 26% of "very good" and "good" risk patients having unfavourable outcomes. Hypercalcaemia has been associated with poor outcomes in several cancers including multiple myeloma, but is not included in current DLBCL prognostic scoring models. Aim To determine the prognostic significance of hypercalcaemia in newly diagnosed DLBCL and identify relationships between hypercalcaemia and other established prognostic variables including cell of origin, and the components of the revised international prognostic index (R-IPI including impaired performance status, older age, advanced stage, elevated lactate dehydrogenase, and extranodal involvement. Methods Retrospective cohort study at two academic healthcare networks in Melbourne, Australia. All patients with a newly diagnosed DLBCL by WHO 2008 criteria were eligible for inclusion. Cases were identified from hospital lymphoma databases. Only cases with adequate clinical information including baseline characteristics, therapy received and outcomes were included. Cell of origin was determined by the modified Hans criteria. At Monash Health, patients were included from September 2002 until December 2015, and from Western Health, patients were included from January 2012 until December 2015. Hypercalcaemia was defined as a serum calcium >2.6mmol/L within 30 days prior to, or 15 days following the diagnosis of DLBCL. Serum calcium levels were corrected for hypoalbuminaemia if present. Immunohistochemical cell of origin was recorded using modified Hans criteria (Meyer et al. 2011). Survival correlates were estimated by Kaplan-Meier log rank method. Multivariable analysis was performed by Cox regression. Relationships between serum calcium and other covariates were examined by t test for continuous variables and chi for categorical variables. This project was approved by the human research review boards at each institution. Results A total of 514 p

The transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) have recently been found to be targeted for destruction by the immunomodulatory drugs (IMiDs). However, the role of Ikaros and Aiolos and why their loss leads to multiple myeloma (MM) cell death remains unclear. We have used CRISPRCas9 genome editing to delete IKZF1 and IKZF3 in human MM cell lines to gain further insight into their downstream gene regulatory networks. Consistent with the action of the IMiDs, loss of either of these genes resulted in both a G1/S cell cycle arrest and induction of apoptosis. This was not dependent on the subsequent reduction of the IRF4-MYC "axis", as neither IRF4 or MYC were consistently downregulated across all cell lines examined (despite cell death occurring) and overexpression of either factor failed to rescue for Ikaros loss. This led us to further investigate the transcriptional changes resulting from deletion of Ikaros, Aiolos or treatment with the IMiD lenalidomide using RNA-sequencing. Supporting their central role in the action of the IMiDs, significant overlap was seen on loss of Ikaros, Aiolos, or lenalidomide treatment; with loss of Ikaros/Aiolos accounting for approximately 75% of differential gene expression following lenalidomide. Importantly both Ikaros and Aiolos were found to repress the expression of interferon stimulated genes (ISGs) and their loss led to the activation of an interferon-like response, possibly contributing to cell death. In keeping with this, treatment with lenalidomide and low dose IFNb resulted in synergistic cell death. Furthermore, CD38 appeared to be both an ISG and repressed target of Ikaros/Aiolos through their interaction with the nucleosome remodelling and deacetylase complex, and its expression is increased at both mRNA and protein level on their loss. Given recent clinical studies have shown improved outcomes with combination treatment with lenalidomide and the anti-CD38 monoclonal antibody daratumumab, we wondered whether th

Introduction Plasmablastic lymphoma (PBL) is a rare, aggressive large cell lymphoma, first described in 1997. PBL is strongly associated with immunodeficient states, such as HIV infection and solid organ transplantation, but up to one third of cases are reported to occur in immunocompetent patients. The pathogenesis of PBL is incompletely understood, though the oncogenic impact of EBV, in particular in the context of dysregulated immune surveillance, together with acquired abnormalities in the MYC pathway appear to play key roles in many cases. Plasma cell markers such as CD138 and CD38 are typically positive, as well as CD30 in a significant subset. Classical B cell markers such as CD20, CD19 and PAX5 are typically absent. The literature on clinical outcomes in PBL is generally limited to small, single-centre case series. Reports describe an aggressive disease of poor prognosis, with median survival of 8 to 15 months, with one series reporting a longer median survival of 32 months. Methods We retrospectively identified patients diagnosed with PBL between 1999 and 2019 from 16 sites across Australia, the United Kingdom and Canada. Patients aged >=18 years with confirmed tissue diagnosis of PBL at their local treating centre were included. Factors associated with overall survival (OS) were analysed using Cox regression, stratified by site to account for heterogeneity across sites. Risk time for mortality began on the date of diagnosis and ended on the date of death. Patients who were alive, lost to follow-up or transferred to another centre for care, were censored on the date of last follow-up. Risk factors analysed included age, year of diagnosis, HIV status, MYC rearrangement status, CD30 status, lactate dehydrogenase level, disease stage by Lugano consensus criteria, and bone marrow involvement. Results We identified 197 patients with PBL (Table 1). The median age at diagnosis was 55 years (range 18-95) and there was a male predominance (69%). 37% of patients were

The transcription factor IRF4 is essential for the survival of both normal plasma cells and the plasma cell malignancy multiple myeloma (MM). However the basis for this dependency remains uncertain, with the prevailing view that IRF4 loss in MM induces "death by a thousand cuts." Here we have explored the genetic basis for IRF4 addiction through CRISPR-Cas9 mediated deletion of IRF4 in three lenalidomide sensitive MM cells lines (MM1S, OPM2 and H929) and one resistant cell line (RPMI-8226). Loss of IRF4 resulted in marked loss of viability, irrespective of lenalidomide sensitivity, which was driven by two distinct mechanisms: 1) induction of apoptosis, which was rescued through prior deletion of apoptotic mediators BAX and BAK, and 2) proliferative arrest, which persisted despite rescue from apoptosis. RNA-sequencing following IRF4 inactivation identified a core group of 86 genes whose dysregulation was shared across all 4 MM cell lines. We identified 31 common down-regulated genes (IRF4 activated), including several with previously described roles in survival (such as KLF2, TNFRSF17/BCMA and MYB). Furthermore, MYC, a previously identified target of IRF4 known to play an important role in MM pathogenesis and survival, was down-regulated in 3 of the 4 cell lines. Most surprisingly, IRF4 inactivation resulted in 55 common up-regulated genes that included two BH3-only pro-apoptotic proteins, BMF and BCL2L11 (BIM). Up-regulation of BMF appeared more marked than BIM with an average fold change of 6.5x (range 2.7 - 10.3) vs 1.9x (range: 1.8 - 2.2) respectively. Remarkably, genetic ablation of BMF in the OPM2 cell line and BMF/BIM in the MM1S largely protected from the cell death observed following IRF4 inactivation, suggesting that IRF4 maintains MM survival through the transcriptional repression of BMF and BIM. Our results confirm the vital role of IRF4 for MM proliferation and survival, and shed further light into its critical downstream transcriptional targets. Most in

Introduction Plasmablastic lymphoma (PBL) is a rare, aggressive large cell lymphoma, first described in 1997. PBL is strongly associated with immunodeficient states, such as HIV infection and solid organ transplantation, but up to one third of cases are reported to occur in immunocompetent patients. The pathogenesis of PBL is incompletely understood, though the oncogenic impact of EBV, in particular in the context of dysregulated immune surveillance, together with acquired abnormalities in the MYC pathway appear to play key roles in many cases. Plasma cell markers such as CD138 and CD38 are typically positive, as well as CD30 in a significant subset. Classical B cell markers such as CD20, CD19 and PAX5 are typically absent. The literature on clinical outcomes in PBL is generally limited to small, single-centre case series. Reports describe an aggressive disease of poor prognosis, with median survival of 8 to 15 months, with one series reporting a longer median survival of 32 months. Methods We retrospectively identified patients diagnosed with PBL between 1999 and 2019 from 16 sites across Australia, the United Kingdom and Canada. Patients aged >=18 years with confirmed tissue diagnosis of PBL at their local treating centre were included. Factors associated with overall survival (OS) were analysed using Cox regression, stratified by site to account for heterogeneity across sites. Risk time for mortality began on the date of diagnosis and ended on the date of death. Patients who were alive, lost to follow-up or transferred to another centre for care, were censored on the date of last follow-up. Risk factors analysed included age, year of diagnosis, HIV status, MYC rearrangement status, CD30 status, lactate dehydrogenase level, disease stage by Lugano consensus criteria, and bone marrow involvement. Results We identified 197 patients with PBL (Table 1). The median age at diagnosis was 55 years (range 18-95) and there was a male predominance (69%). 37% of patients were

The transcription factor IRF4 is essential for the survival of both normal plasma cells and the plasma cell malignancy multiple myeloma (MM). However the basis for this dependency remains uncertain, with the prevailing view that IRF4 loss in MM induces "death by a thousand cuts." Here we have explored the genetic basis for IRF4 addiction through CRISPR-Cas9 mediated deletion of IRF4 in three lenalidomide sensitive MM cells lines (MM1S, OPM2 and H929) and one resistant cell line (RPMI-8226). Loss of IRF4 resulted in marked loss of viability, irrespective of lenalidomide sensitivity, which was driven by two distinct mechanisms: 1) induction of apoptosis, which was rescued through prior deletion of apoptotic mediators BAX and BAK, and 2) proliferative arrest, which persisted despite rescue from apoptosis. RNA-sequencing following IRF4 inactivation identified a core group of 86 genes whose dysregulation was shared across all 4 MM cell lines. We identified 31 common down-regulated genes (IRF4 activated), including several with previously described roles in survival (such as KLF2, TNFRSF17/BCMA and MYB). Furthermore, MYC, a previously identified target of IRF4 known to play an important role in MM pathogenesis and survival, was down-regulated in 3 of the 4 cell lines. Most surprisingly, IRF4 inactivation resulted in 55 common up-regulated genes that included two BH3-only pro-apoptotic proteins, BMF and BCL2L11 (BIM). Up-regulation of BMF appeared more marked than BIM with an average fold change of 6.5x (range 2.7 - 10.3) vs 1.9x (range: 1.8 - 2.2) respectively. Remarkably, genetic ablation of BMF in the OPM2 cell line and BMF/BIM in the MM1S largely protected from the cell death observed following IRF4 inactivation, suggesting that IRF4 maintains MM survival through the transcriptional repression of BMF and BIM. Our results confirm the vital role of IRF4 for MM proliferation and survival, and shed further light into its critical downstream transcriptional targets. Most in