Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Insight into the mechanism of CD34+ cell mobilisation impairment in multiple myeloma patients treated with anti-CD38 therapy.

Induction therapy followed by CD34+ cell mobilisation and autologous transplantation represents standard of care for multiple myeloma (MM). However, the anti-CD38 monoclonal antibodies daratumumab and isatuximab have been associated with mobilisation impairment, yet the mechanism remains unclear. In this study, we investigated the effect of three different regimens (dara-VCd, isa-KRd and VTd) on CD34+ cells using flow cytometry and transcriptomics. Decreased CD34+ cell peak concentration and yields, longer collection and delayed engraftment were reproduced after dara-VCd/isa-KRd versus VTd induction in 34 patients in total. Using flow cytometry, we detected major changes in the proportion of apheresis product and bone marrow CD34+ subsets in patients treated with regimens containing anti-CD38 therapy; however, without any decrease in CD38high B-lymphoid progenitors in both materials. RNA-seq of mobilised CD34+ cells from 21 patients showed that adhesion genes are overexpressed in CD34+ cells after dara-VCd/isa-KRd and JCAD, NRP2, MDK, ITGA3 and CLEC3B were identified as potential target genes. Finally, direct in vitro effect of isatuximab in upregulating JCAD and CLEC3B was confirmed by quantitative PCR. These findings suggest that upregulated adhesion-related interactions, rather than killing of CD34+ cells by effector mechanisms, could be leading causes of decreased mobilisation efficacy in MM patients treated with anti-CD38 therapy.

Transcriptome Analysis of Diffuse Large B-Cell Lymphoma Cells Inducibly Expressing MyD88 L265P Mutation Identifies Upregulated CD44, LGALS3, NFKBIZ, and BATF as Downstream Targets of Oncogenic NF-κB Signaling.

During innate immune responses, myeloid differentiation primary response 88 (MyD88) functions as a critical signaling adaptor protein integrating stimuli from toll-like receptors (TLR) and the interleukin-1 receptor (IL-1R) family and translates them into specific cellular outcomes. In B cells, somatic mutations in MyD88 trigger oncogenic NF-κB signaling independent of receptor stimulation, which leads to the development of B-cell malignancies. However, the exact molecular mechanisms and downstream signaling targets remain unresolved. We established an inducible system to introduce MyD88 to lymphoma cell lines and performed transcriptomic analysis (RNA-seq) to identify genes differentially expressed by MyD88 bearing the L265P oncogenic mutation. We show that MyD88L265P activates NF-κB signaling and upregulates genes that might contribute to lymphomagenesis, including CD44, LGALS3 (coding Galectin-3), NFKBIZ (coding IkBƺ), and BATF. Moreover, we demonstrate that CD44 can serve as a marker of the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) and that CD44 expression is correlated with overall survival in DLBCL patients. Our results shed new light on the downstream outcomes of MyD88L265P oncogenic signaling that might be involved in cellular transformation and provide novel therapeutical targets.

The deubiquitinase OTUD1 regulates immunoglobulin production and proteasome inhibitor sensitivity in multiple myeloma.

Serum monoclonal immunoglobulin (Ig) is the main diagnostic factor for patients with multiple myeloma (MM), however its prognostic potential remains unclear. On a large MM patient cohort (n = 4146), we observe no correlation between serum Ig levels and patient survival, while amount of intracellular Ig has a strong predictive effect. Focused CRISPR screen, transcriptional and proteomic analysis identify deubiquitinase OTUD1 as a critical mediator of Ig synthesis, proteasome inhibitor sensitivity and tumor burden in MM. Mechanistically, OTUD1 deubiquitinates peroxiredoxin 4 (PRDX4), protecting it from endoplasmic reticulum (ER)-associated degradation. In turn, PRDX4 facilitates Ig production which coincides with the accumulation of unfolded proteins and higher ER stress. The elevated load on proteasome ultimately potentiates myeloma response to proteasome inhibitors providing a window for a rational therapy. Collectively, our findings support the significance of the Ig production machinery as a biomarker and target in the combinatory treatment of MM patients.

Deubiquitinase OTUD1 Resolves Stalled Translation on polyA and Rare Codon Rich mRNAs.

OTUD1 is a deubiquitinating enzyme involved in many cellular processes including cancer and innate, immune signaling pathways. Here, we perform a proximity labeling-based interactome study that identifies OTUD1 largely present in the translation and RNA metabolism protein complexes. Biochemical analysis validates OTUD1 association with ribosome subunits, elongation factors and the E3 ubiquitin ligase ZNF598 but not with the translation initiation machinery. OTUD1 catalytic activity suppresses polyA triggered ribosome stalling through inhibition of ZNF598-mediated RPS10 ubiquitination and stimulates formation of polysomes. Finally, analysis of gene expression suggests that OTUD1 regulates the stability of rare codon rich mRNAs by antagonizing ZNF598.

Modeling hypoxia facilitates cancer cell survival through downregulation of p53 expression.

A hypoxic environment of rapidly growing tumor cells makes them resistant to antitumor drugs. Mimicking hypoxia with iron chelator deferoxamine, suppressed cell death induced by widely used anticancer drugs doxorubicin or cisplatin. Deferoxamine decreased the number of dead (detached) cells, the size of SubG1 population, the release of cytochrome c, and the processing of caspase-3 in HCT116 colon carcinoma cells treated with cisplatin or doxorubicin. Deferoxamine-mediated suppression of apoptosis correlated with the level of pro-apoptotic Bcl-2 family proteins Bax, Bid, and Puma, which stimulate mitochondrial apoptotic pathway through permeabilization of the outer mitochondrial membrane and cytochrome c release. Here we show that one of the reasons for apoptosis suppression is downregulation of p53 expression under hypoxic conditions, and, as a result, attenuation of the expression of pro-apoptotic Bcl-2 family proteins. Indeed, p53 knock-out did not affect the stabilization of hypoxia-inducible factor but made undetectable the expression of pro-apoptotic proteins.

Transcriptional profiling of circulating tumor cells in multiple myeloma: a new model to understand disease dissemination.

The reason why a few myeloma cells egress from the bone marrow (BM) into peripheral blood (PB) remains unknown. Here, we investigated molecular hallmarks of circulating tumor cells (CTCs) to identify the events leading to myeloma trafficking into the bloodstream. After using next-generation flow to isolate matched CTCs and BM tumor cells from 32 patients, we found high correlation in gene expression at single-cell and bulk levels (r ≥ 0.94, P = 10-16), with only 55 genes differentially expressed between CTCs and BM tumor cells. CTCs overexpressed genes involved in inflammation, hypoxia, or epithelial-mesenchymal transition, whereas genes related with proliferation were downregulated in CTCs. The cancer stem cell marker CD44 was overexpressed in CTCs, and its knockdown significantly reduced migration of MM cells towards SDF1-α and their adhesion to fibronectin. Approximately half (29/55) of genes differentially expressed in CTCs were prognostic in patients with newly-diagnosed myeloma (n = 553; CoMMpass). In a multivariate analysis including the R-ISS, overexpression of CENPF and LGALS1 was significantly associated with inferior survival. Altogether, these results help understanding the presence of CTCs in PB and suggest that hypoxic BM niches together with a pro-inflammatory microenvironment induce an arrest in proliferation, forcing tumor cells to circulate in PB and seek other BM niches to continue growing.

Targeting mitochondria by α-tocopheryl succinate overcomes hypoxia-mediated tumor cell resistance to treatment.

Rapidly proliferating tumor cells easily become hypoxic. This results in acquired stability towards treatment with anticancer drugs. Here, we show that cells grown at 0.1 % oxygen are more resistant towards treatment with the conventionally used anticancer drugs doxorubicin and cisplatin. The stimulation of apoptosis, as assessed by the number of cells in the SubG1 fraction of the cell cycle, release of cytochrome c into the cytosol, activation of caspase-3, and cleavage of PARP, was markedly suppressed under low oxygen content or when hypoxia was mimicked by deferoxamine. Hypoxia or deferoxamine treatment was accompanied by stabilization of the hypoxia-inducible factor (HIF-1). The downregulation of HIF-1 using siRNA technique restored cell sensitivity to treatment under hypoxic conditions to the levels detected under normoxic conditions. In contrast to cisplatin or doxorubicin, α-tocopheryl succinate (α-TOS), a compound that targets mitochondria, stimulated cell death irrespective of the oxygen concentration. Moreover, under hypoxic condition cell death induced by α-TOS was even enhanced. Thus, α-TOS can successfully overcome resistance to treatment caused by hypoxia, which makes α-TOS an attractive candidate for antitumor therapy via mitochondrial targeting.

Conformational heterogeneity of methyl 4-hydroxycinnamate: a gas-phase UV-IR spectroscopic study.

UV excitation and IR absorption spectroscopy on jet-cooled molecules is used to study the conformational heterogeneity of methyl 4-hydroxycinnamate, a model chromophore of the Photoactive Yellow Protein (PYP), and to determine the spectroscopic properties of the various conformers. UV-UV depletion spectroscopy identifies four different species with distinct electronic excitation spectra. Quantum chemical calculations argue that these species are associated with different conformers involving the s-cis/s-trans configuration of the ester with respect to the propenyl C-C single bond and the syn/anti orientation of the phenolic OH group. IR-UV hole-burning spectroscopy is used to record their IR absorption spectra in the fingerprint region. Comparison with IR absorption spectra predicted by quantum chemical calculations provides vibrational markers for each of the conformers, on the basis of which each of the species observed with UV-UV depletion spectroscopy is assigned. Although both DFT and wave function methods reproduce experimental frequencies, we find that calculations at the MP2 level are necessary to obtain agreement with experimentally observed intensities. To elucidate the role of the environment, we compare the IR spectra of the isolated conformers with IR spectra of methyl 4-hydroxycinnamate-water clusters, and with IR spectra of methyl 4-hydroxycinnamate in solution.

High-resolution excitation and absorption spectroscopy of gas-phase p-coumaric acid: unveiling an elusive chromophore.

We report on the first successful high-resolution spectroscopic studies on isolated para-coumaric acid, the chromophore of the photoactive yellow protein which has become a model system for studying biological light-induced signal transduction. Employing various double-resonance multiphoton ionization techniques in combination with mass-resolved ion detection and the results of quantum chemical calculations, we identify three conformations the molecule can adopt under our experimental conditions. The vibrational activity in the excitation spectra allows us to conclude that in the Franck-Condon region accessed from the ground state S(1) is the V'(pipi*) state. Interestingly, we find considerable out-of-plane vibrational activity, indicating that the molecule adopts a nonplanar geometry in S(1). The ionization requirements show that after excitation rapid internal conversion takes place to a lower-lying npi* state. Such a state has been postulated by ab initio calculations on para-coumaric acid and derivatives, but until the present study no direct evidence had been found for its presence.