Systems medicine dissection of chr1q-amp reveals a novel PBX1-FOXM1 axis for targeted therapy in multiple myeloma.

Understanding the biological and clinical impact of copy number aberrations (CNAs) on the development of precision therapies in cancer remains an unmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNA conferring an adverse prognosis in several types of cancer, including in the blood cancer multiple myeloma (MM). Although several genes across chromosome 1 (chr1q) portend high-risk MM disease, the underpinning molecular etiology remains elusive. Here, with reference to the 3-dimensional (3D) chromatin structure, we integrate multi-omics data sets from patients with MM with genetic variables to obtain an associated clinical risk map across chr1q and to identify 103 adverse prognosis genes in chr1q-amp MM. Prominent among these genes, the transcription factor PBX1 is ectopically expressed by genetic amplification and epigenetic activation of its own preserved 3D regulatory domain. By binding to reprogrammed superenhancers, PBX1 directly regulates critical oncogenic pathways and a FOXM1-dependent transcriptional program. Together, PBX1 and FOXM1 activate a proliferative gene signature that predicts adverse prognosis across multiple types of cancer. Notably, pharmacological disruption of the PBX1-FOXM1 axis with existing agents (thiostrepton) and a novel PBX1 small molecule inhibitor (T417) is selectively toxic against chr1q-amp myeloma and solid tumor cells. Overall, our systems medicine approach successfully identifies CNA-driven oncogenic circuitries, links them to clinical phenotypes, and proposes novel CNA-targeted therapy strategies in MM and other types of cancer.

Systems level profiling of chemotherapy-induced stress resolution in cancer cells reveals druggable trade-offs.

Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization.

Non-crystalline light chain proximal tubulopathy, a morphologically protean entity.

BACKGROUND: Light chain proximal tubulopathy (LCPT) is a rare form of paraprotein-related disease, occurring in two main histopathological forms: crystalline and non-crystalline. The clinicopathological features, treatment strategies and outcomes, especially of the non-crystalline form, are not well described. METHODS: We conducted a single-centre retrospective case series of 12 LCPT patients, 5 crystalline and 7 non-crystalline, between 2005 and 2021. RESULTS: The median age was 69.5 years (range 47-80). Ten patients presented with CKD and significant proteinuria (median estimated glomerular filtration rate of 43.5 ml/min/1.73 m2; urine protein:creatinine ratio 328 mg/mmol). Only six patients had known haematological disease at the time of renal biopsy. Multiple myeloma (MM) was diagnosed in seven patients cases and monoclonal gammopathy of renal significance (MGRS) in five patients. A clone was detected in all cases combining serum/urine electrophoresis and free light chain (LC) assays. Crystalline and non-crystalline variants had similar clinical presentations. For the non-crystalline variant, a diagnosis was reached based on a combination of CKD without another cause, haematological workup, LC restriction on immunofluorescence and abnormalities on electron microscopy (EM). Nine of 12 patients received clone-directed treatment. Patients who achieved haematological response (including all non-crystalline LCPT) had improved renal outcomes over a median follow-up of 79 months. CONCLUSIONS: The non-crystalline variant may go unrecognised because of its subtle histopathological features and requires EM to distinguish it from 'excessive LC resorption without tubular injury'. Clone-directed treatment with good haematological response improves renal outcomes in both variants but limited data exist in MGRS. Multicentre prospective studies are needed to better define the clinicopathological characteristics associated with poor outcomes and optimize treatment strategies in patients with MGRS.

The innate sensor ZBP1-IRF3 axis regulates cell proliferation in multiple myeloma.

Multiple myeloma is a malignancy of plasma cells initiated and driven by primary and secondary genetic events. However, myeloma plasma cell survival and proliferation might be sustained by non-genetic drivers. Z-DNA-binding protein 1 (ZBP1; also known as DAI) is an interferon-inducible, Z-nucleic acid sensor that triggers RIPK3-MLKL-mediated necroptosis in mice. ZBP1 also interacts with TBK1 and the transcription factor IRF3 but the function of this interaction is unclear, and the role of the ZBP1-IRF3 axis in cancer is not known. Here we show that ZBP1 is selectively expressed in late B-cell development in both human and murine cells and it is required for optimal T-cell-dependent humoral immune responses. In myeloma plasma cells, the interaction of constitutively expressed ZBP1 with TBK1 and IRF3 results in IRF3 phosphorylation. IRF3 directly binds and activates cell cycle genes, in part through co-operation with the plasma cell lineage-defining transcription factor IRF4, thereby promoting myeloma cell proliferation. This generates a novel, potentially therapeutically targetable and relatively selective myeloma cell addiction to the ZBP1-IRF3 axis. Our data also show a noncanonical function of constitutive ZBP1 in human cells and expand our knowledge of the role of cellular immune sensors in cancer biology.

Tazemetostat for patients with relapsed or refractory follicular lymphoma: an open-label, single-arm, multicentre, phase 2 trial.

BACKGROUND: Activating mutations of EZH2, an epigenetic regulator, are present in approximately 20% of patients with follicular lymphoma. We investigated the activity and safety of tazemetostat, a first-in-class, oral EZH2 inhibitor, in patients with follicular lymphoma. METHODS: This study was an open-label, single-arm, phase 2 trial done at 38 clinics or hospitals in France, the UK, Australia, Canada, Poland, Italy, Ukraine, Germany, and the USA. Eligible patients were adults (≥18 years) with histologically confirmed follicular lymphoma (grade 1, 2, 3a, or 3b) that had relapsed or was refractory to two or more systemic therapies, had an Eastern Cooperative Oncology Group performance status of 0-2, and had sufficient tumour tissue for central testing of EZH2 mutation status. Patients were categorised by EZH2 status: mutant (EZH2mut) or wild-type (EZH2WT). Patients received 800 mg of tazemetostat orally twice per day in continuous 28-day cycles. The primary endpoint was objective response rate based on the 2007 International Working Group criteria for non-Hodgkin lymphoma, assessed by an independent radiology committee. Activity and safety analyses were done in patients who received one dose or more of tazemetostat. This study is registered with ClinicalTrials.gov, NCT01897571, and follow-up is ongoing. FINDINGS: Between July 9, 2015, and May 24, 2019, 99 patients (45 in the EZH2mut cohort and 54 in the EZH2WT cohort) were enrolled in the study. At data cutoff for the analysis (Aug 9, 2019), the median follow-up was 22·0 months (IQR 12·0-26·7) for the EZH2mut cohort and 35·9 months (24·9-40·5) for the EZH2WT cohort. The objective response rate was 69% (95% CI 53-82; 31 of 45 patients) in the EZH2mut cohort and 35% (23-49; 19 of 54 patients) in the EZH2WT cohort. Median duration of response was 10·9 months (95% CI 7·2-not estimable [NE]) in the EZH2mut cohort and 13·0 months (5·6-NE) in the EZH2WT cohort; median progression-free survival was 13·8 months (10·7-22·0) and 11·1 months (3·7-14·6). Among all 99 patients, treatment-related grade 3 or worse adverse events included thrombocytopenia (three [3%]), neutropenia (three [3%]), and anaemia (two [2%]). Serious treatment-related adverse events were reported in four (4%) of 99 patients. There were no treatment-related deaths. INTERPRETATION: Tazemetostat monotherapy showed clinically meaningful, durable responses and was generally well tolerated in heavily pretreated patients with relapsed or refractory follicular lymphoma. Tazemetostat is a novel treatment for patients with follicular lymphoma. FUNDING: Epizyme.

High resolution IgH repertoire analysis reveals fetal liver as the likely origin of life-long, innate B lymphopoiesis in humans.

The ontogeny of the natural, public IgM repertoire remains incompletely explored. Here, high-resolution immunogenetic analysis of B cells from (unrelated) fetal, child, and adult samples, shows that although fetal liver (FL) and bone marrow (FBM) IgM repertoires are equally diversified, FL is the main source of IgM natural immunity during the 2nd trimester. Strikingly, 0.25% of all prenatal clonotypes, comprising 18.7% of the expressed repertoire, are shared with the postnatal samples, consistent with persisting fetal IgM+ B cells being a source of natural IgM repertoire in adult life. Further, the origins of specific stereotypic IgM+ B cell receptors associated with chronic lymphocytic leukemia, can be traced back to fetal B cell lymphopoiesis, suggesting that persisting fetal B cells can be subject to malignant transformation late in life. Overall, these novel data provide unique insights into the ontogeny of physiological and malignant B lymphopoiesis that spans the human lifetime.

Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs.

Diamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin(-)CD34(+)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(-)CD36(-) bone marrow cells as EEP giving rise to BFU-E, and Lin(-)CD34(+/-)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(+)CD36(+) cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis.

Results of a multicentre UK-wide retrospective study evaluating the efficacy of pixantrone in relapsed, refractory diffuse large B cell lymphoma.

Relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) in those unfit or ineligible for autologous stem cell transplantation is associated with a poor outcome and new treatment approaches are needed. Pixantrone is a novel aza-anthracenedione which is structurally similar to anthracyclines and is licenced in R/R DLBCL and National Institute for Health and Care Excellence (NICE)-approved following the PIX301 trial. No data exist post-NICE approval. We performed a UK-wide retrospective multi-centre study of 92 R/R DLBCL who received pixantrone. Eighty-five per cent had refractory disease and 72% had an international prognostic index (IPI) 3-5 at commencement of pixantrone. The median progression-free survival (PFS) was 2·0 months (95% confidence interval (CI) 1·5-2·4) and the median overall survival was 3·4 months (95% CI 2·7-4·5). The overall response rate was 24% (complete response 10%; partial response 14%). We demonstrate that pixantrone has limited activity in a cohort of high risk, predominantly refractory DLBCL. Multivariate Cox regression revealed that patients who relapsed >12 months after first line treatment, those with fewer prior lines of therapy and relapsed (non-refractory) DLBCL had improved PFS. The major population of unmet need are those with refractory DLBCL who are poorly represented within trials and in whom pixantrone appears less efficacious compared to relapsed DLBCL.

Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762.

The bromodomain and extraterminal (BET) protein BRD2-4 inhibitors hold therapeutic promise in preclinical models of hematologic malignancies. However, translation of these data to molecules suitable for clinical development has yet to be accomplished. Herein we expand the mechanistic understanding of BET inhibitors in multiple myeloma by using the chemical probe molecule I-BET151. I-BET151 induces apoptosis and exerts strong antiproliferative effect in vitro and in vivo. This is associated with contrasting effects on oncogenic MYC and HEXIM1, an inhibitor of the transcriptional activator P-TEFb. I-BET151 causes transcriptional repression of MYC and MYC-dependent programs by abrogating recruitment to the chromatin of the P-TEFb component CDK9 in a BRD2-4-dependent manner. In contrast, transcriptional upregulation of HEXIM1 is BRD2-4 independent. Finally, preclinical studies show that I-BET762 has a favorable pharmacologic profile as an oral agent and that it inhibits myeloma cell proliferation, resulting in survival advantage in a systemic myeloma xenograft model. These data provide a strong rationale for extending the clinical testing of the novel antimyeloma agent I-BET762 and reveal insights into biologic pathways required for myeloma cell proliferation.

Myeloma Propagating Cells, Drug Resistance and Relapse.

Multiple myeloma (MM) is an incurable tumor of the plasma cells, the terminally differentiated immunoglobulin secreting B lineage cells. The genetic make-up of MM has been extensively characterized but its impact on the biology of the disease is incomplete without more precise knowledge of the identity and functional role of cells with multiple myeloma propagating activity (MMPA). We review here recent data that link MMPA with myeloma clonotypic populations organized in a cellular hierarchy that mirrors normal B cell development and also with drug resistance and disease relapse. We further propose a conceptual framework which, with optimal use of recent technological advances in genomics and phenomics, could allow dissection of the cellular and molecular properties of cells with MMPA, drug resistance and in vivo relapse in an integrated and patient-specific manner. There is real hope that these approaches will significantly contribute to further improvements in disease control, overall survival, and possibly cure of patients with MM.

Glycosylphosphatidylinositol-specific, CD1d-restricted T cells in paroxysmal nocturnal hemoglobinuria.

The mechanism of bone marrow failure (BMF) in paroxysmal nocturnal hemoglobinuria (PNH) is not yet known. Because in PNH the biosynthesis of the glycolipid molecule glycosylphosphatidylinositol (GPI) is disrupted in hematopoietic stem and progenitor cells by a somatic mutation in the PIG-A gene, BMF might result from an autoimmune attack, whereby T cells target GPI in normal cells, whereas PIG-A mutant GPI-negative cells are spared. In a deliberate test of this hypothesis, we have demonstrated in PNH patients the presence of CD8(+) T cells reactive against antigen-presenting cells (APCs) loaded with GPI. These T cells were significantly more abundant in PNH patients than in healthy controls; their reactivity depended on CD1d expression and they increased upon coculture with CD1d-expressing, GPI-positive APCs. In GPI-specific T cells captured by CD1d dimer technology, we identified, through global T-cell receptor α (TCRα) analysis, an invariant TCRVα21 sequence, which was then found at frequencies higher than background in the TCR repertoire of 6 of 11 PNH patients. Thus, a novel, autoreactive, CD1d-restricted, GPI-specific T-cell population, enriched in an invariant TCRα chain, is expanded in PNH patients and may be responsible for BMF in PNH.

Tyrosine kinase inhibitors impair B-cell immune responses in CML through off-target inhibition of kinases important for cell signaling.

Tyrosine kinase inhibitors (TKIs) have significant off-target multikinase inhibitory effects. We aimed to study the impact of TKIs on the in vivo B-cell response to vaccination. Cellular and humoral responses to influenza and pneumococcal vaccines were evaluated in 51 chronic phase chronic myeloid leukemia (CML) patients on imatinib, or second-line dasatinib and nilotinib, and 24 controls. Following vaccination, CML patients on TKI had significant impairment of IgM humoral response to pneumococcus compared with controls (IgM titer 79.0 vs 200 U/mL, P = .0006), associated with significantly lower frequencies of peripheral blood IgM memory B cells. To elucidate whether CML itself or treatment with TKI was responsible for the impaired humoral response, we assessed memory B-cell subsets in paired samples collected before and after imatinib therapy. Treatment with imatinib was associated with significant reductions in IgM memory B cells. In vitro coincubation of B cells with plasma from CML patients on TKI or with imatinib, dasatinib, or nilotinib induced significant and dose-dependent inhibition of Bruton's tyrosine kinase and indirectly its downstream substrate, phospholipase-C-γ2, both important in B-cell signaling and survival. These data indicate that TKIs, through off-target inhibition of kinases important in B-cell signaling, reduce memory B-cell frequencies and induce significant impairment of B-cell responses in CML.

Clinical drug resistance linked to interconvertible phenotypic and functional states of tumor-propagating cells in multiple myeloma.

The phenotype and function of cells enriched in tumor-propagating activity and their relationship to the phenotypic architecture in multiple myeloma (MM) are controversial. Here, in a cohort of 30 patients, we show that MM composes 4 hierarchically organized, clonally related subpopulations, which, although phenotypically distinct, share the same oncogenic chromosomal abnormalities as well as immunoglobulin heavy chain complementarity region 3 area sequence. Assessed in xenograft assays, myeloma-propagating activity is the exclusive property of a population characterized by its ability for bidirectional transition between the dominant CD19(-)CD138(+) plasma cell (PC) and a low frequency CD19(-)CD138(-) subpopulation (termed Pre-PC); in addition, Pre-PCs are more quiescent and unlike PCs, are primarily localized at extramedullary sites. As shown by gene expression profiling, compared with PCs, Pre-PCs are enriched in epigenetic regulators, suggesting that epigenetic plasticity underpins the phenotypic diversification of myeloma-propagating cells. Prospective assessment in paired, pretreatment, and posttreatment bone marrow samples shows that Pre-PCs are up to 300-fold more drug-resistant than PCs. Thus, clinical drug resistance in MM is linked to reversible, bidirectional phenotypic transition of myeloma-propagating cells. These novel biologic insights have important clinical implications in relation to assessment of minimal residual disease and development of alternative therapeutic strategies in MM.

Perturbation of fetal liver hematopoietic stem and progenitor cell development by trisomy 21.

The 40-fold increase in childhood megakaryocyte-erythroid and B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in perturbing fetal hematopoiesis. Here, we show that compared with primary disomic controls, primary T21 fetal liver (FL) hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors are markedly increased, whereas granulocyte-macrophage progenitors are reduced. Commensurately, HSC and megakaryocyte-erythroid progenitors show higher clonogenicity, with increased megakaryocyte, megakaryocyte-erythroid, and replatable blast colonies. Biased megakaryocyte-erythroid-primed gene expression was detected as early as the HSC compartment. In lymphopoiesis, T21 FL lymphoid-primed multipotential progenitors and early lymphoid progenitor numbers are maintained, but there was a 10-fold reduction in committed PreproB-lymphoid progenitors and the functional B-cell potential of HSC and early lymphoid progenitor is severely impaired, in tandem with reduced early lymphoid gene expression. The same pattern was seen in all T21 FL samples and no samples had GATA1 mutations. Therefore, T21 itself causes multiple distinct defects in FL myelo- and lymphopoiesis.

Graft invariant natural killer T-cell dose predicts risk of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation.

Invariant natural killer T (iNKT) cells are powerful immunomodulatory cells that in mice regulate a variety of immune responses, including acute GVHD (aGVHD). However, their clinical relevance and in particular their role in clinical aGVHD are not known. We studied whether peripheral blood stem cell (PBSC) graft iNKT-cell dose affects on the occurrence of clinically significant grade II-IV aGVHD in patients (n = 57) undergoing sibling, HLA-identical allogeneic HSCT. In multivariate analysis, CD4(-) iNKT-cell dose was the only graft parameter to predict clinically significant aGVHD. The cumulative incidence of grade II-IV aGVHD in patients receiving CD4(-) iNKT-cell doses above and below the median were 24.2% and 71.4%, respectively (P = .0008); low CD4(-) iNKT-cell dose was associated with a relative risk of grade II-IV aGVHD of 4.27 (P = .0023; 95% CI, 1.68-10.85). Consistent with a role of iNKT cells in regulating aGVHD, in mixed lymphocyte reaction assays, CD4(-) iNKT cells effectively suppressed T-cell proliferation and IFN-γ secretion in a contact-dependent manner. In conclusion, higher doses of CD4(-) iNKT cells in PBSC grafts are associated with protection from aGVHD. This effect could be harnessed for prevention of aGVHD.