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BCMA targeting chimeric antigen receptor (CAR) T cell therapy has shown deep and durable responses in multiple myeloma. However, relapse following therapy is frequently observed, and mechanisms of resistance remain ill-defined. Here, we perform single cell genomic characterization of longitudinal samples from a patient who relapsed after initial CAR T cell treatment with lack of response to retreatment. We report selection, following initial CAR T cell infusion, of a clone with biallelic loss of BCMA acquired by deletion of one allele and a mutation that creates an early stop codon on the second allele. This loss leads to lack of CAR T cell proliferation following the second infusion and is reflected by lack of soluble BCMA in patient serum. Our analysis suggests the need for careful detection of BCMA gene alterations in multiple myeloma cells from relapse following CAR T cell therapy. Relapse following BCMA targeted CAR T-cell therapy is frequently observed in patients with multiple myeloma (MM). Here, by single cell transcriptome profiling on serially collected bone marrow samples, the authors report biallelic loss of BCMA as the mechanism of resistance underlying both relapse and lack of response to a second CAR T infusion in a patient with MM.

Alternative cleavage and polyadenylation (ApA) is known to alter untranslated region (3ʹUTR) length but can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the coding region. We analyzed 46 3ʹ-seq and RNA-seq profiles from normal human tissues, primary immune cells, and multiple myeloma (MM) samples and created an atlas of 4927 high-confidence IpA events represented in these cell types. IpA isoforms are widely expressed in immune cells, differentially used during B-cell development or in different cellular environments, and can generate truncated proteins lacking C-terminal functional domains. This can mimic ectodomain shedding through loss of transmembrane domains or alter the binding specificity of proteins with DNA-binding or protein–protein interaction domains. MM cells display a striking loss of IpA isoforms expressed in plasma cells, associated with shorter progression-free survival and impacting key genes in MM biology and response to lenalidomide. Recognition of intronic polyadenylation (IpA) signals can lead to expression of truncated proteins lacking C terminal domains. Analysis of 3ʹ -seq and RNA-seq shows that IpA is widespread in circulating immune cells, while multiple myeloma cells show loss of IpA isoforms that are normally expressed in plasma cells, impacting key genes in the disease.

PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D. In this work, the authors report a sophisticated combination of genetic, biophysical, and biochemical analyses to identifies the cycling conformational states of PPM1D. The findings reveal how an allosteric inhibitor locks the protein into a conformationally inactive state, and explain the distribution of PPM1D activating mutations in cancer.

DNA topoisomerases are believed to promote transcription by removing excessive DNA supercoils produced during elongation. However, it is unclear how topoisomerases in eukaryotes are recruited and function in the transcription pathway in the context of nucleosomes. To address this problem we present high-resolution genome-wide maps of one of the major eukaryotic topoisomerases, Topoisomerase II (Top2) and nucleosomes in the budding yeast, Saccharomyces cerevisiae. Our data indicate that at promoters Top2 binds primarily to DNA that is nucleosome-free. However, although nucleosome loss enables Top2 occupancy, the opposite is not the case and the loss of Top2 has little effect on nucleosome density. We also find that Top2 is involved in transcription. Not only is Top2 enriched at highly transcribed genes, but Top2 is required redundantly with Top1 for optimal recruitment of RNA polymerase II at their promoters. These findings and the examination of candidate-activated genes suggest that nucleosome loss induced by nucleosome remodeling factors during gene activation enables Top2 binding, which in turn acts redundantly with Top1 to enhance recruitment of RNA polymerase II.

Key Points Myelodysplastic syndrome (MDS) is one of the most common haematological malignancies and is associated with increased age and exposure to previous chemotherapy and radiation. It is characterized by cytopenias, morphological dysplasia and a propensity to transform to acute myeloid leukaemia (AML). Clonal haematopoiesis of indeterminate potential (CHIP) is a condition in which a substantial percentage of haematopoietic cells bear a somatic mutation in a gene that is recurrently mutated in haematological malignancies, including MDS. CHIP is strongly associated with age and an increased risk of haematological malignancy. More than 50 recurrently mutated genes have been identified in MDS, many of which occur in genes encoding RNA splicing factors, epigenetic regulators, haematopoietic transcription factors and kinase signalling pathways. Individual mutations in MDS are associated with specific morphological findings, have independent prognostic significance and can predict response to therapy in some cases. AML that arises out of a pre-existing MDS can be distinguished from de novo AML by the presence of specific mutations, such as those in splicing factors and certain epigenetic regulators. Some mutations are associated with increased sensitivity or resistance to standard therapeutic interventions, providing new targets for the development of novel therapeutic agents. Currently, allogeneic haematopoietic stem cell transplantation is the only known curative treatment for MDS. This Review discusses the molecular processes and clonal evolution that lead to myelodysplastic syndrome (MDS) and secondary acute myeloid leukaemia, highlighting the ways in which these insights are shaping the clinical management of MDS. Myelodysplastic syndrome (MDS) is a clonal disease that arises from the expansion of mutated haematopoietic stem cells. In a spectrum of myeloid disorders ranging from clonal haematopoiesis of indeterminate potential (CHIP) to secondary acute myeloid leukaemia (sAML), MDS is distinguished by the presence of peripheral blood cytopenias, dysplastic haematopoietic differentiation and the absence of features that define acute leukaemia. More than 50 recurrently mutated genes are involved in the pathogenesis of MDS, including genes that encode proteins involved in pre-mRNA splicing, epigenetic regulation and transcription. In this Review we discuss the molecular processes that lead to CHIP and further clonal evolution to MDS and sAML. We also highlight the ways in which these insights are shaping the clinical management of MDS, including classification schemata, prognostic scoring systems and therapeutic approaches.

For decades, anticancer targeted therapies have been designed to inhibit kinases or other enzyme classes and have profoundly benefited many patients. However, novel approaches are required to target transcription factors, scaffolding proteins and other proteins central to cancer biology that typically lack catalytic activity and have remained mostly recalcitrant to drug development. The selective degradation of target proteins is an attractive approach to expand the druggable proteome, and the selective oestrogen receptor degrader fulvestrant served as an early example of this concept. Following a long and tragic history in the clinic, the immunomodulatory imide drug (IMiD) thalidomide was discovered to exert its therapeutic activity via a novel and unexpected mechanism of action: targeting proteins to an E3 ubiquitin ligase for subsequent proteasomal degradation. This discovery has paralleled and directly catalysed myriad breakthroughs in drug development, leading to the rapid maturation of generalizable chemical platforms for the targeted degradation of previously undruggable proteins. Decades of clinical experience have established front-line roles for thalidomide analogues, including lenalidomide and pomalidomide, in the treatment of haematological malignancies. With a new generation of ‘degrader’ drugs currently in development, this experience provides crucial insights into class-wide features of degraders, including a unique pharmacology, mechanisms of resistance and emerging therapeutic opportunities. Herein, we review these past experiences and discuss their application in the clinical development of novel degrader therapies.The discovery that the anticancer activity of thalidomide and its analogues, such as lenalidomide, reflects drug-induced degradation of specific target proteins has heightened interest in novel ‘degrader’ drugs. Herein, the authors review the wide and expanding use of thalidomide analogues in the treatment of multiple cancers and outline how lessons learned from this experience, particularly with lenalidomide, can guide the clinical development of new targeted protein degradation platforms.

BackgroundChimeric antigen receptor (CAR) T-cell therapy has demonstrated significant benefits in the treatment of relapsed/refractory multiple myeloma (RRMM). However, these outcomes can be compromised by severe complications, including cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) and immune effector cell-associated hematotoxicity (ICAHT), predisposing for life-threatening infections.MethodsThis retrospective observational study examined a total of 129 patients with RRMM who had received idecabtagene vicleucel (ide-cel) at two major myeloma centers in Germany and one center in the USA to assess the Endothelial Activation and Stress Index (EASIX) as a risk marker for an unfavorable clinical course and outcome after CAR T-cell therapy. EASIX is calculated by lactate dehydrogenase (U/L) × creatinine (mg/dL) / platelets (109 cells/L) and was determined before lymphodepletion (baseline) and at the day of CAR T-cell infusion (day 0). The analysis was extended to EASIX derivatives and the CAR-HEMATOTOX score.ResultsAn elevated baseline EASIX (>median) was identified as a risk marker for severe late ICAHT, manifesting with an impaired hematopoietic reconstitution and pronounced cytopenias during the late post-CAR-T period. Patients with high EASIX levels (>upper quartile) were particularly at risk, as evidenced by an increased rate of an aplastic phenotype of neutrophil recovery, severe late-onset infections and ICANS. Finally, we found associations between baseline EASIX and an inferior progression-free and overall survival. Moreover, the EASIX at day 0 also demonstrated potential to serve as a risk marker for post-CAR-T complications and adverse outcomes.ConclusionsIn conclusion, EASIX aids in risk stratification at clinically relevant time points prior to CAR T-cell therapy with ide-cel. Increased EASIX levels might help clinicians to identify vulnerable patients to adapt peri-CAR-T management at an early stage.

In budding yeast, telomeres and the mating type (HM) loci are found in a heterochromatin-like silent structure initiated by Rap1 and extended by the interaction of Silencing Information Regulator (Sir) proteins with histones. Binding data demonstrate that both the H3 and H4 N-terminal domains required for silencing in vivo interact directly with Sir3 and Sir4 in vitro. The role of H4 lysine 16 deacetylation is well established in Sir3 protein recruitment; however, that of the H3 N-terminal tail has remained unclear. To characterize the role of H3 in silent chromatin formation and compare it to H4 we have generated comprehensive high resolution genome-wide binding maps of heterochromatin proteins. We found that H4 lysine 16 deacetylation is required for the recruitment and spreading of heterochromatin proteins at all telomeres and HM loci. In contrast, the H3 N terminus is required for neither recruitment nor spreading of Sir proteins. Instead, deletion of the H3 tail leads to increased accessibility within heterochromatin of an ectopic bacterial dam methylase and the decreased mobility of an HML heterochromatic fragment in sucrose gradients. These findings indicate an altered chromatin structure. We propose that Sir proteins recruited by the H4 tail then interact with the H3 tail to form a higher order silent chromatin structure.

Despite the widespread use of carfilzomib (K) in relapsed/refractory multiple myeloma (RRMM), there is no consensus on optimal K dose in milligrams per square meter (mg/m2) or dosing schedule. We assessed three modern K prescribing patterns in RRMM using a large United States electronic health record-derived database. Our final cohort ( n  = 486) included 136 patients (28.0%) who received K 56 mg/m2 once weekly (K56-1x), 86 (17.7%) who received 56 mg/m2 twice weekly (K56-2x), and 264 (54.3%) who received 70 mg/m2 once weekly (K70-1x). Between 2016 and 2023, once-weekly dosing became more common: K70-1x proportions changed from 21.1% in 2016 to 50.6% in 2023, K56-1x from 15.8% to 37.0%, and K56-2x from 63.2% to 12.3%. Median progression-free survival was 13.0 months [95% confidence interval (CI) 11.2-20.7] for K56-1x, 13.2 months (95% CI 9.0-28.1 months) for K56-2x, and 10.9 months (95% CI 9.9-15.3 months) for K70-1x; these differences were not statistically significant (log-rank p  = 0.46). Rates of heart failure was comparable (<5% in all cohorts). In summary, our findings do not support improved outcomes with twice-weekly carfilzomib in RRMM. K56-1x may provide the best balance of efficacy, safety, and avoidance of time toxicity from frequent infusions.

Selinexor is an orally bioavailable selective inhibitor of nuclear export compound that inhibits exportin‐1 (XPO1), a novel therapeutic target that is overexpressed in multiple myeloma (MM) and is responsible for the transport of ∼220 nuclear proteins to the cytoplasm, including tumour suppressor proteins. Inhibition of this process has demonstrated substantial antimyeloma activity in preclinical studies, both alone and in combination with established MM therapeutics. Based on a clinical trial programme encompassing multiple combination regimens, selinexor‐based therapy has been approved for the treatment of relapsed/refractory MM (RRMM), with selinexor‐dexamethasone approved in the later‐relapse setting for penta‐refractory patients and selinexor‐bortezomib‐dexamethasone approved for patients who have received ≥1 prior therapy. Here, we provide a comprehensive review of the clinical data on selinexor‐based regimens, including recent updates from the 2022 American Society of Hematology annual meeting, and summarise ongoing studies of this novel targeted agent in newly diagnosed MM and RRMM.