T cell characteristics impact response and resistance to T cell-redirecting bispecific antibodies in multiple myeloma.

PURPOSE: Bispecific antibodies (BsAbs) directed against B-cell maturation antigen (BCMA; teclistamab) or the orphan G protein-coupled receptor GPRC5D (talquetamab) induce deep and durable responses in heavily pretreated MM patients. However, mechanisms underlying primary and acquired resistance remain poorly understood. EXPERIMENTAL DESIGN: The anti-MM activity of teclistamab and talquetamab was evaluated in bone marrow (BM) samples from MM patients. T-cell phenotype and function were assessed in BM/peripheral blood samples obtained from MM patients who were treated with these BsAbs. RESULTS: In ex vivo killing assays with 41 BM samples from BsAb-naïve MM patients, teclistamab- and talquetamab-mediated MM lysis were strongly correlated (r=0.73, P<0.0001). Both BsAbs exhibited poor activity in samples with high regulatory T-cell (Treg) numbers and a low T-cell/MM cell-ratio. Furthermore, comprehensive phenotyping of BM samples derived from patients treated with teclistamab or talquetamab, revealed that high frequencies of PD-1+ CD4+ T-cells, CTLA4+ CD4+ T-cells, and CD38+ CD4+ T-cells were associated with primary resistance. Although this lack of response was linked to modest increase in expression of inhibitory receptors, increasing T-cell/MM cell-ratios by adding extra T-cells enhanced sensitivity to BsAbs. Further, treatment with BsAbs resulted in an increased proportion of T-cells expressing exhaustion markers (PD-1, TIGIT, and TIM-3), which was accompanied by reduced T-cell proliferative potential and cytokine secretion, as well as impaired anti-tumor efficacy in ex vivo experiments. CONCLUSIONS: Primary resistance is characterized by a low T-cell/MM cell-ratio and Treg-driven immunosuppression, while reduced T-cell fitness due to continuous BsAb-mediated T-cell activation may contribute to development of acquired resistance.

Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-mediated Antitumor Responses In Vivo.

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.

Minimal Residual Disease in Myeloma: Application for Clinical Care and New Drug Registration.

The development of novel agents has transformed the treatment paradigm for multiple myeloma, with minimal residual disease (MRD) negativity now achievable across the entire disease spectrum. Bone marrow-based technologies to assess MRD, including approaches using next-generation flow and next-generation sequencing, have provided real-time clinical tools for the sensitive detection and monitoring of MRD in patients with multiple myeloma. Complementary liquid biopsy-based assays are now quickly progressing with some, such as mass spectrometry methods, being very close to clinical use, while others utilizing nucleic acid-based technologies are still developing and will prove important to further our understanding of the biology of MRD. On the regulatory front, multiple retrospective individual patient and clinical trial level meta-analyses have already shown and will continue to assess the potential of MRD as a surrogate for patient outcome. Given all this progress, it is not surprising that a number of clinicians are now considering using MRD to inform real-world clinical care of patients across the spectrum from smoldering myeloma to relapsed refractory multiple myeloma, with each disease setting presenting key challenges and questions that will need to be addressed through clinical trials. The pace of advances in targeted and immune therapies in multiple myeloma is unprecedented, and novel MRD-driven biomarker strategies are essential to accelerate innovative clinical trials leading to regulatory approval of novel treatments and continued improvement in patient outcomes.

Belantamab mafodotin in combination with novel agents in relapsed/refractory multiple myeloma: DREAMM-5 study design.

Belantamab mafodotin (belamaf) is a BCMA-targeted antibody-drug conjugate recently approved as monotherapy for adults with relapsed/refractory multiple myeloma who have received ≥4 prior therapies. Belamaf binds to BCMA and eliminates myeloma cells by multimodal mechanisms of action. The cytotoxic and potential immunomodulatory properties of belamaf have led to novel combination studies with other anticancer therapies. Here, we describe the rationale and design of DREAMM-5, an ongoing Phase I/II platform study evaluating the safety and efficacy of belamaf combined with novel agents, including GSK3174998 (OX40 agonist), feladilimab (an ICOS; GSK3359609), nirogacestat (a gamma-secretase inhibitor; PF-03084014) and dostarlimab (a PD-1 blocker) versus belamaf monotherapy for patients with relapsed/refractory multiple myeloma. Clinical trial registration: NCT04126200 (ClinicalTrials.gov).

Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

Activation of the p53-MDM4 regulatory axis defines the anti-tumour response to PRMT5 inhibition through its role in regulating cellular splicing.

Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.

The histone chaperone HJURP is a new independent prognostic marker for luminal A breast carcinoma.

BACKGROUND: Breast cancer is a heterogeneous disease with different molecular subtypes that have varying responses to therapy. An ongoing challenge in breast cancer research is to distinguish high-risk patients from good prognosis patients. This is particularly difficult in the low-grade, ER-positive luminal A tumors, where robust diagnostic tools to aid clinical treatment decisions are lacking. Recent data implicating chromatin regulators in cancer initiation and progression offers a promising avenue to develop new tools to help guide clinical decisions. METHODS: Here we exploit a published transcriptome dataset and an independent validation cohort to correlate the mRNA expression of selected chromatin regulators with respect to the four intrinsic breast cancer molecular subtypes. We then perform univariate and multivariate analyses to compare the prognostic value of a panel of chromatin regulators to Ki67, a currently utilized proliferation marker. RESULTS: Unsupervised hierarchical clustering revealed a gene cluster containing several histone chaperones and histone variants highly-expressed in the proliferative subtypes (basal-like, HER2-positive, luminal B) but not in the luminal A subtype. Several chromatin regulators, including the histone chaperones CAF-1 (subunits p150 and p60), ASF1b, and HJURP, and the centromeric histone variant CENP-A, associated with local and metastatic relapse and poor patient outcome. Importantly, we find that HJURP can discriminate favorable and unfavorable outcome within the luminal A subtype, outperforming the currently utilized proliferation marker Ki67, as an independent prognostic marker for luminal A patients. CONCLUSIONS: The integration of chromatin regulators as clinical biomarkers, in particular the histone chaperone HJURP, will help guide patient substratification and treatment options for low-risk luminal A breast carcinoma patients.

Phosphorylation and DNA binding of HJURP determine its centromeric recruitment and function in CenH3(CENP-A) loading.

Centromeres, epigenetically defined by the presence of the histone H3 variant CenH3, are essential for ensuring proper chromosome segregation. In mammals, centromeric CenH3(CENP-A) deposition requires its dedicated chaperone HJURP and occurs during telophase/early G1. We find that the cell-cycle-dependent recruitment of HJURP to centromeres depends on its timely phosphorylation controlled via cyclin-dependent kinases. A nonphosphorylatable HJURP mutant localizes prematurely to centromeres in S and G2 phase. This unregulated targeting causes a premature loading of CenH3(CENP-A) at centromeres, and cell-cycle delays ensue. Once recruited to centromeres, HJURP functions to promote CenH3(CENP-A) deposition by a mechanism involving a unique DNA-binding domain. With our findings, we propose a model wherein (1) the phosphorylation state of HJURP controls its centromeric recruitment in a cell-cycle-dependent manner, and (2) HJURP binding to DNA is a mechanistic determinant in CenH3(CENP-A) loading.

The survival gene MED4 explains low penetrance retinoblastoma in patients with large RB1 deletion.

Retinoblastoma is a non-hereditary as well as an inherited pediatric tumor of the developing retina resulting from the inactivation of both copies of the RB1 tumor suppressor gene. Familial retinoblastoma is a highly penetrant genetic disease that usually develops by carrying germline mutations that inactivate one allele of the RB1 gene, leading to multiple retinoblastomas. However, large and complete germline RB1 deletions are associated with low or no tumor risk for reasons that remain unknown. In this study, we define a minimal genomic region associated with this low penetrance. This region encompasses few genes including MED4 a subunit of the mediator complex. We further show that retinoblastoma RB1 -/- cells cannot survive in the absence of MED4, both in vitro and in orthotopic xenograft models in vivo, therefore identifying MED4 as a survival gene in retinoblastoma. We propose that the contiguous loss of the adjacent retinoblastoma gene, MED4, explains the low penetrance in patients with large deletions that include both RB1 and MED4. Our findings also point to another synthetic lethal target in tumors with inactivated RB1 and highlight the importance of collateral damage in carcinogenesis.

Barrier-to-Autointegration Factor influences specific histone modifications.

Defects in the nuclear envelope or nuclear 'lamina' networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells.

Cell cycle dynamics of histone variants at the centromere, a model for chromosomal landmarks.

Classical heterochromatin chromosomal landmarks, such as centromeres and telomeres, are characterized by specific chromatin signatures. Among these, the incorporation of histone variants has recently emerged as an important feature. Using the centromere as a paradigm, we consider the role of histone variant dynamics in locus-specific chromatin organization. We describe the distinct location and dynamics of CenH3, H3.3, and H2AZ at the centromere during the cell cycle. This leads us to present the current view concerning modes of incorporation at this chromosomal landmark. Finally, we highlight the importance of histone variants in the crosstalk between centric and pericentric domains for maintaining centromere identity.

SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin.

HP1 enrichment at pericentric heterochromatin is considered important for centromere function. Although HP1 binding to H3K9me3 can explain its accumulation at pericentric heterochromatin, how it is initially targeted there remains unclear. Here, in mouse cells, we reveal the presence of long nuclear noncoding transcripts corresponding to major satellite repeats at the periphery of pericentric heterochromatin. Furthermore, we find that major transcripts in the forward orientation specifically associate with SUMO-modified HP1 proteins. We identified this modification as SUMO-1 and mapped it in the hinge domain of HP1α. Notably, the hinge domain and its SUMOylation proved critical to promote the initial targeting of HP1α to pericentric domains using de novo localization assays, whereas they are dispensable for maintenance of HP1 domains. We propose that SUMO-HP1, through a specific association with major forward transcript, is guided at the pericentric heterochromatin domain to seed further HP1 localization.

Barrier-to-autointegration factor proteome reveals chromatin-regulatory partners.

Nuclear lamin filaments and associated proteins form a nucleoskeletal ("lamina") network required for transcription, replication, chromatin organization and epigenetic regulation in metazoans. Lamina defects cause human disease ("laminopathies") and are linked to aging. Barrier-to-autointegration factor (BAF) is a mobile and essential component of the nuclear lamina that binds directly to histones, lamins and LEM-domain proteins, including the inner nuclear membrane protein emerin, and has roles in chromatin structure, mitosis and gene regulation. To understand BAF's mechanisms of action, BAF associated proteins were affinity-purified from HeLa cell nuclear lysates using BAF-conjugated beads, and identified by tandem mass spectrometry or independently identified and quantified using the iTRAQ method. We recovered A- and B-type lamins and core histones, all known to bind BAF directly, plus four human transcription factors (Requiem, NonO, p15, LEDGF), disease-linked proteins (e.g., Huntingtin, Treacle) and several proteins and enzymes that regulate chromatin. Association with endogenous BAF was independently validated by co-immunoprecipitation from HeLa cells for seven candidates including Requiem, poly(ADP-ribose) polymerase 1 (PARP1), retinoblastoma binding protein 4 (RBBP4), damage-specific DNA binding protein 1 (DDB1) and DDB2. Interestingly, endogenous BAF and emerin each associated with DDB2 and CUL4A in a UV- and time-dependent manner, suggesting BAF and emerin have dynamic roles in genome integrity and might help couple DNA damage responses to the nuclear lamina network. We conclude this proteome is a rich source of candidate partners for BAF and potentially also A- and B-type lamins, which may reveal how chromatin regulation and genome integrity are linked to nuclear structure.

Binding of barrier to autointegration factor (BAF) to histone H3 and selected linker histones including H1.1.

Barrier to autointegration factor (BAF) is an essential conserved double-stranded DNA-binding protein in metazoans. BAF binds directly to LEM domain nuclear proteins (e.g. LAP2, Emerin, and MAN1), lamin A, homeodomain transcription factors, and human immunodeficiency virus type 1-encoded proteins. BAF influences higher order chromatin structure and is required to assemble nuclei. BAF also facilitates retroviral preintegration complex insertion into target DNA in vitro, through unknown mechanisms. We report that BAF binds directly and selectively to linker histone H1.1 (among three subtypes tested) and core histone H3 with affinities of approximately 700 nm and approximately 100-200 nm, respectively, in vitro and in vivo. Mutations at the bottom and top surfaces of the BAF dimer disrupted or enhanced, respectively, this binding and affected H1 and H3 similarly. Biochemical studies showed that C-terminal residues 108-215 of histone H1.1 and the N-terminal tail plus helix alphaN in the core of histone H3.1 were each necessary and sufficient to bind BAF. Based on its interactions with histones and DNA, we propose BAF might bind nucleosomes in vivo.

Nuclear membrane protein emerin: roles in gene regulation, actin dynamics and human disease.

Loss of emerin, a nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy (EDMD), characterized by muscle weakening, contractures of major tendons and potentially lethal cardiac conduction system defects. Emerin has a LEM-domain and therefore binds barrier-to-autointegration factor (BAF), a conserved chromatin protein essential for cell division. BAF recruits emerin to chromatin and regulates higher-order chromatin structure during nuclear assembly. Emerin also binds filaments formed by A-type lamins, mutations in which also cause EDMD. Other partners for emerin include nesprin-1alpha and transcriptional regulators such as germ cell-less (GCL). The binding affinities of these partners range from 4nM (nesprin-1alpha) to 200 nM (BAF), and are physiologically significant. Biochemical studies therefore provide a valid means to predict the properties of emerin-lamin complexes in vivo. Emerin and lamin A together form stable complexes with either BAF or GCL in vitro. BAF, however, competes with GCL for binding to emerin in vitro. These and additional partners, notably actin and nuclear myosin II, suggest disease-relevant roles for emerin in gene regulation and the mechanical interity of the nucleus.

Regulated interaction of the Fanconi anemia protein, FANCD2, with chromatin.

DNA damage activates the monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, resulting in the assembly of FANCD2 nuclear foci. In the current study, we characterize structural features of FANCD2 required for this intranuclear translocation. We have previously identified 2 normal mRNA splice variants of FANCD2, one containing exon 44 sequence at the 3' end (FANCD2-44) and one containing exon 43 sequence (FANCD2-43). The 2 predicted FANCD2 proteins differ in their carboxy terminal 24 amino acids. In stably transfected FANCD2(-/-) fibroblasts, FANCD2-44 and FANCD2-43 proteins were monoubiquitinated on K561. Only FANCD2-44 corrected the mitomycin C (MMC) sensitivity of the transfected cells. We find that monoubiquitinated FANCD2-44 was translocated from the soluble nuclear compartment into chromatin. A mutant form of FANCD2-44 (FANCD2-K561R) was not monoubiquitinated and failed to bind chromatin. A truncated FANCD2 protein (Exon44-T), lacking the carboxy terminal 24 amino acids encoded by exon 44 but retaining K561, and another mutant FANCD2 protein, with a single amino acid substitution at a conserved residue within the C-terminal 24 amino acids (D1428A), were monoubiquitinated. Both mutants were targeted to chromatin but failed to correct MMC sensitivity. Taken together, our results indicate that monoubiquitination of FANCD2 regulates chromatin binding and that D1428 within the carboxy terminal acidic sequence encoded by exon 44 is independently required for functional complementation of FA-D2 cells. We hypothesize that the carboxy terminus of FANCD2-44 plays a critical role in sensing or repairing DNA damage.

Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours.

Activation of the Hedgehog (Hh) signalling pathway by sporadic mutations or in familial conditions such as Gorlin's syndrome is associated with tumorigenesis in skin, the cerebellum and skeletal muscle. Here we show that a wide range of digestive tract tumours, including most of those originating in the oesophagus, stomach, biliary tract and pancreas, but not in the colon, display increased Hh pathway activity, which is suppressible by cyclopamine, a Hh pathway antagonist. Cyclopamine also suppresses cell growth in vitro and causes durable regression of xenograft tumours in vivo. Unlike in Gorlin's syndrome tumours, pathway activity and cell growth in these digestive tract tumours are driven by endogenous expression of Hh ligands, as indicated by the presence of Sonic hedgehog and Indian hedgehog transcripts, by the pathway- and growth-inhibitory activity of a Hh-neutralizing antibody, and by the dramatic growth-stimulatory activity of exogenously added Hh ligand. Our results identify a group of common lethal malignancies in which Hh pathway activity, essential for tumour growth, is activated not by mutation but by ligand expression.

A novel diagnostic screen for defects in the Fanconi anemia pathway.

Fanconi anemia (FA) is an autosomal recessive chromosomal instability syndrome characterized by congenital abnormalities, progressive bone marrow failure, and cancer predisposition. Although patients with FA are candidates for bone marrow transplantation or gene therapy, their phenotypic heterogeneity can delay or obscure diagnosis. The current diagnostic test for FA consists of cytogenetic quantitation of chromosomal breakage in response to diepoxybutane (DEB) or mitomycin C (MMC). Recent studies have elucidated a biochemical pathway for Fanconi anemia that culminates in the monoubiquitination of the FANCD2 protein. In the current study, we develop a new rapid diagnostic and subtyping FA assay amenable for screening broad populations at risk of FA. Primary lymphocytes were assayed for FANCD2 monoubiquitination by immunoblot. The absence of the monoubiquitinated FANCD2 isoform correlated with the diagnosis of FA by DEB testing in 11 known patients with FA, 37 patients referred for possible FA, and 29 healthy control subjects. Monoubiquitination of FANCD2 was normal in other bone marrow failure syndromes and chromosomal breakage syndromes. A combination of retroviral gene transfer and FANCD2 immunoblotting provides a rapid subtyping assay for patients newly diagnosed with FA. These new FA screening assays would allow efficient testing of broad populations at risk.

Repercusiones psicológicas de la ausencia testicular y el uso de prótesis / Psychological repercussions of the absence of testis and the use of prosthesis

Se presenta la experiencia con 17 niños en quienes se implantó una prótesis testicular por diversas causas y la repercusión psicológica de esta intervención que tienen los pacientes, sobre todo los mayores de 12 años y los padres