Correcting PCR amplification errors in unique molecular identifiers to generate accurate numbers of sequencing molecules.

Unique molecular identifiers are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are a source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing unique molecular identifiers using homotrimeric nucleotide blocks provides an error-correcting solution that allows absolute counting of sequenced molecules.

Determinants of durable humoral and T cell immunity in myeloma patients following COVID-19 vaccination.

OBJECTIVE: To describe determinants of persisting humoral and cellular immune response to the second COVID-19 vaccination among patients with myeloma. METHODS: This is a prospective, observational study utilising the RUDYstudy.org platform. Participants reported their second and third COVID-19 vaccination dates. Myeloma patients had an Anti-S antibody level sample taken at least 21 days after their second vaccination and a repeat sample before their third vaccination. RESULTS: 60 patients provided samples at least 3 weeks (median 57.5 days) after their second vaccination and before their third vaccination (median 176.0 days after second vaccine dose). Low Anti-S antibody levels (<50 IU/mL) doubled during this interval (p = .023) and, in the 47 participants with T-spot data, there was a 25% increase negative T-spot tests (p = .008). Low anti-S antibody levels prior to the third vaccination were predicted by lower Anti-S antibody level and negative T-spot status after the second vaccine. Independent determinants of a negative T-spot included increasing age, previous COVID infection, high CD4 count and lower percentage change in Anti-S antibody levels. CONCLUSIONS: Negative T-spot results predict low Anti-S antibody levels (<50 IU/mL) following a second COVID-19 vaccination and a number of biomarkers predict T cell responses in myeloma patients.

The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms.

The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.

Iberdomide increases innate and adaptive immune cell subsets in the bone marrow of patients with relapsed/refractory multiple myeloma.

Iberdomide is a potent cereblon E3 ligase modulator (CELMoD agent) with promising efficacy and safety as a monotherapy or in combination with other therapies in patients with relapsed/refractory multiple myeloma (RRMM). Using a custom mass cytometry panel designed for large-scale immunophenotyping of the bone marrow tumor microenvironment (TME), we demonstrate significant increases of effector T and natural killer (NK) cells in a cohort of 93 patients with multiple myeloma (MM) treated with iberdomide, correlating findings to disease characteristics, prior therapy, and a peripheral blood immune phenotype. Notably, changes are dose dependent, associated with objective response, and independent of prior refractoriness to MM therapies. This suggests that iberdomide broadly induces innate and adaptive immune activation in the TME, contributing to its antitumor efficacy. Our approach establishes a strategy to study treatment-induced changes in the TME of patients with MM and, more broadly, patients with cancer and establishes rational combination strategies for iberdomide with immune-enhancing therapies to treat MM.

PRMT5 inhibition shows in vitro efficacy against H3K27M-altered diffuse midline glioma, but does not extend survival in vivo.

H3K27-altered Diffuse Midline Glioma (DMG) is a universally fatal paediatric brainstem tumour. The prevalent driver mutation H3K27M creates a unique epigenetic landscape that may also establish therapeutic vulnerabilities to epigenetic inhibitors. However, while HDAC, EZH2 and BET inhibitors have proven somewhat effective in pre-clinical models, none have translated into clinical benefit due to either poor blood-brain barrier penetration, lack of efficacy or toxicity. Thus, there remains an urgent need for new DMG treatments. Here, we performed wider screening of an epigenetic inhibitor library and identified inhibitors of protein arginine methyltransferases (PRMTs) among the top hits reducing DMG cell viability. Two of the most effective inhibitors, LLY-283 and GSK591, were targeted against PRMT5 using distinct binding mechanisms and reduced the viability of a subset of DMG cells expressing wild-type TP53 and mutant ACVR1. RNA-sequencing and phenotypic analyses revealed that LLY-283 could reduce the viability, clonogenicity and invasion of DMG cells in vitro, representing three clinically important phenotypes, but failed to prolong survival in an orthotopic xenograft model. Together, these data show the challenges of DMG treatment and highlight PRMT5 inhibitors for consideration in future studies of combination treatments.

Discovery of a Potent, Selective, and Cell-Active SPIN1 Inhibitor.

The methyl-lysine reader protein SPIN1 plays important roles in various human diseases. However, targeting methyl-lysine reader proteins has been challenging. Very few cellularly active SPIN1 inhibitors have been developed. We previously reported that our G9a/GLP inhibitor UNC0638 weakly inhibited SPIN1. Here, we present our comprehensive structure-activity relationship study that led to the discovery of compound 11, a dual SPIN1 and G9a/GLP inhibitor, and compound 18 (MS8535), a SPIN1 selective inhibitor. We solved the cocrystal structure of SPIN1 in complex with 11, confirming that 11 occupied one of the three Tudor domains. Importantly, 18 displayed high selectivity for SPIN1 over 38 epigenetic targets, including G9a/GLP, and concentration dependently disrupted the interactions of SPIN1 and H3 in cells. Furthermore, 18 was bioavailable in mice. We also developed 19 (MS8535N), which was inactive against SPIN1, as a negative control of 18. Collectively, these compounds are useful chemical tools to study biological functions of SPIN1.