Proteasome Inhibitor Research Articles

HOP stabilizes the HSFA1a and plays a main role in the onset of thermomorphogenesis.

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1(HSFA1) family. Members of this family interact with heat shock protein70(HSP70) and heat shock protein 90(HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role ofcochaperones, such as the HSP70-HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown.Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a-responsive-responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability.

Marizomib (Salinosporamide A) Promotes Apoptosis in A375 and G361 Melanoma Cancer Cells

Malignant melanoma—a tumor originating from melanocytes—is characterized by dynamic growth and frequent metastases in the early stage of development. Current therapy methods are still insufficient, and there is a need to search for new ways of treating this malady. The induction of apoptosis—physiological cell death—by proteasome inhibitors is recognized as an effective method of non-invasive elimination of cancer cells. In our research, we wanted to check the potential of marizomib (MZB, salinosporamide A, NPI-0052)—an irreversible proteasome inhibitor derived from the marine actinomycete Salinispora tropica—to induce apoptosis in A375 and G361 malignant melanoma cells. We determined the cytotoxic activity of marizomib by performing an MTT test. Ethidium bromide and acridine orange staining demonstrated the disruption of membrane integrity in the examined cell lines. We confirmed the proapoptotic activity of marizomib by flow cytometry with the use of an FITC-Annexin V assay. A Western blot analysis presented an increase in the expression of proteins related to endoplasmic reticulum (ER) stress as well as markers of the apoptosis. The gathered findings suggest that marizomib induced the ER stress in the examined melanoma cancer cells and directed them towards the apoptosis pathway.

Gastrodin Improves the Activity of the Ubiquitin–Proteasome System and the Autophagy–Lysosome Pathway to Degrade Mutant Huntingtin

Gastrodin (GAS) is the main chemical component of the traditional Chinese herb Gastrodia elata (called “Tianma” in Chinese), which has been used to treat neurological conditions, including headaches, epilepsy, stroke, and memory loss. To our knowledge, it is unclear whether GAS has a therapeutic effect on Huntington’s disease (HD). In the present study, we evaluated the effect of GAS on the degradation of mutant huntingtin protein (mHtt) by using PC12 cells transfected with N-terminal mHtt Q74. We found that 0.1–100 μM GAS had no effect on the survival rate of Q23 and Q74 PC12 cells after 24–48 h of incubation. The ubiquitin–proteasome system (UPS) is the main system that clears misfolded proteins in eukaryotic cells. Mutated Htt significantly upregulated total ubiquitinated protein (Ub) expression, decreased chymotrypsin-like, trypsin-like and caspase-like peptidase activity, and reduced the colocalization of the 20S proteasome with mHtt. GAS (25 μM) attenuated all of the abovementioned pathological changes, and the regulatory effect of GAS on mHtt was found to be abolished by MG132, a proteasome inhibitor. The autophagy–lysosome pathway (ALP) is another system for misfolded protein degradation. Although GAS downregulated the expression of autophagy markers (LC3II and P62), it increased the colocalization of LC3II with lysosomal associated membrane protein 1 (LAMP1), which indicates that ALP was activated. Moreover, GAS prevented mHtt-induced neuronal damage in PC12 cells. GAS has a selective effect on mHtt in Q74 PC12 cells and has no effect on Q23 and proteins encoded by other genes containing long CAGs, such as Rbm33 (10 CAG repeats) and Hcn1 (>30 CAG repeats). Furthermore, oral administration of 100 mg/kg GAS increased grip strength and attenuated mHtt aggregates in B6-hHTT130-N transgenic mice. This is a high dose (100 mg/kg GAS) when compared with experiments on HD mice with other small molecules. We will design more doses to evaluate the dose–response relationship of the inhibition effect of GAS on mHtt in our next study. In summary, GAS can promote the degradation of mHtt by activating the UPS and ALP, making it a potential therapeutic agent for HD.

Ixazomib in POEMS syndrome: 'Ixa'ctly what we need?

The role of the proteasome inhibitor ixazomib in the treatment of POEMS syndrome continues to evolve. He and colleagues present the results of a study investigating ixazomib in combination with cyclophosphamide and dexamethasone in newly diagnosed POEMS patients. The triplet showed excellent efficacy and tolerability, and constitutes an effective treatment option for patients with POEMS. Commentary on: He etal. An open-label, prospective trial to evaluate the efficacy and safety of ixazomib in combination with cyclophosphamide and dexamethasone in patients with newly-diagnosed POEMS syndrome. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19626.

Synergistic induction of mitotic pyroptosis and tumor remission by inhibiting proteasome and WEE family kinases

Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.

Multiple myeloma survival in New South Wales, Australia, by treatment era to 2020.

Australia has relatively high multiple myeloma (MM) incidence and mortality rates. Advancements in MM treatment over recent decades have driven improvements in MM survival in high-income countries; however, reporting in Australia is limited. We investigated temporal trends in population-wide MM survival across 3 periods of treatment advancements in New South Wales (NSW), Australia. Individuals with an MM diagnosis in the NSW Cancer Registry between 1985 and 2015 with vital follow-up to 2020, were categorized into 3 previously defined treatment eras according to their diagnosis date (1985-1995, chemotherapy only; 1996-2007, autologous stem cell transplantation; and 2008-2015, novel agents including proteasome inhibitors and immunomodulatory drugs). Both relative survival and cause-specific survival according to Fine and Gray's competing risks cumulative incidence function were calculated by treatment era and age at diagnosis. Overall, 11,591 individuals were included in the study, with a median age of 70 years at diagnosis. Five-year relative survival improved over the 36-year (1985-2020) study period (31.0% in 1985-1995; 41.9% in 1996-2007; and 56.1% in 2008-2015). For individuals diagnosed before 70 years of age, the 5-year relative survival nearly doubled, from 36.5% in 1985-1995 to 68.5% in 2008-2015. Improvements for those > 70 years of age were less pronounced between 1985-1995 and 1996-2007; however, significant improvements were observed for those diagnosed in 2008-2015. Similar overall and age-specific patterns were observed for cause-specific survival. After adjustment for gender and age at diagnosis, treatment era was strongly associated with both relative and cause-specific survival (P < 0.0001). Survival of individuals with MM is improving in Australia with treatment advances. However, older age groups continue to experience poor survival outcomes with only modest improvements over time. Given the increasing prevalence of MM in Australia, the effects of MM treatment on quality of life, particularly in older age, warrant further attention.

Mutations in nucleotide metabolism genes bypass proteasome defects in png-1/NGLY1-deficient Caenorhabditis elegans.

The conserved SKN-1A/Nrf1 transcription factor regulates the expression of proteasome subunit genes and is essential for maintenance of adequate proteasome function in animal development, aging, and stress responses. Unusual among transcription factors, SKN-1A/Nrf1 is a glycoprotein synthesized in the endoplasmic reticulum (ER). N-glycosylated SKN-1A/Nrf1 exits the ER and is deglycosylated in the cytosol by the PNG-1/NGLY1 peptide:N-glycanase. Deglycosylation edits the protein sequence of SKN-1A/Nrf1 by converting N-glycosylated asparagine residues to aspartate, which is necessary for SKN-1A/Nrf1 transcriptional activation of proteasome subunit genes. Homozygous loss-of-function mutations in the peptide:N-glycanase (NGLY1) gene cause NGLY1 deficiency, a congenital disorder of deglycosylation. There are no effective treatments for NGLY1 deficiency. Since SKN-1A/Nrf1 is a major client of NGLY1, the resulting proteasome deficit contributes to NGLY1 disease. We sought to identify targets for mitigation of proteasome dysfunction in NGLY1 deficiency that might indicate new avenues for treatment. We isolated mutations that suppress the sensitivity to proteasome inhibitors caused by inactivation of the NGLY1 ortholog PNG-1 in Caenorhabditis elegans. We identified multiple suppressor mutations affecting 3 conserved genes: rsks-1, tald-1, and ent-4. We show that the suppressors act through a SKN-1/Nrf-independent mechanism and confer proteostasis benefits consistent with amelioration of proteasome dysfunction. ent-4 encodes an intestinal nucleoside/nucleotide transporter, and we show that restriction of nucleotide availability is beneficial, whereas a nucleotide-rich diet exacerbates proteasome dysfunction in PNG-1/NGLY1-deficient C. elegans. Our findings suggest that dietary or pharmacological interventions altering nucleotide availability have the potential to mitigate proteasome insufficiency in NGLY1 deficiency and other diseases associated with proteasome dysfunction.

Mapping the IMiD-dependent cereblon interactome using BioID-proximity labelling.

Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs.

Proteasome and PARP1 dual-target inhibitor for multiple myeloma: Fluzoparib

One of the current mainstream treatments for multiple myeloma (MM) is chemotherapy. However, due to the high clonal heterogeneity and genomic complexity of MM, single-target drugs have limited efficacy and are prone to drug resistance. Therefore, there is an urgent need to develop multi-target drugs against MM. We screened drugs that simultaneously inhibit poly(ADP-ribose) polymerase 1 (PARP1) and 20S proteasome through computer-aided drug discovery (CADD) techniques, and explored the binding mode and dynamic stability of selected inhibitor to proteasome through Molecular biology (MD) simulation method. Thus, the dual-target inhibition effect of fluzoparib was proposed for the first time, and the ability of dual-target inhibition and tumor killing was explored at the enzyme, cell and animal level, respectively. This provides a theoretical and experimental basis for exploring multi-target inhibitory drugs for cancers.

Novel Approaches to Managing Patients with Relapsed and Refractory Waldenström Macroglobulinemia.

Waldenström macroglobulinemia is a rare non-Hodgkin lymphoma (NHL) characterized by lymphoplasmacytic bone marrow infiltration associated with an immunoglobulin M (IgM) monoclonal gammopathy. Over the past two decades, a number of important novel therapies have emerged for the treatment of relapsed and refractory (R/R) WM. The purpose of this review is to discuss these novel agents. Chemoimmunotherapy which formed the basis treatment for R/R WM is slowly being replaced by novel targeted agents. These therapies, including Bruton's tyrosine kinase inhibitors, proteasome inhibitors, and B-cell lymphoma 2 inhibitors, have widened the landscape of management. Emerging therapies currently under investigation, such as bispecific T-cell engagers, chimeric antigen T-cell receptor therapy, and novel small molecule inhibitors, have additionally shown the potential to improve response and survival. The treatment of R/R WM has greatly evolved, in large part due to a greater understanding of the biology of WM, and the evaluation of novel targeted agents in the basket trials of NHL, showing early activity in the small WM cohorts. Combination regimens with these established and emerging novel therapies have the potential to further improve disease control and induce higher rates of deep responses. Strategies aimed at altering the disease trajectory would require randomized controlled trials to provide relevant data on optimal integration and sequencing of more effective and tolerable regimens earlier in the disease course.

HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer.

Resistance to chemotherapy is a major problem in the treatment of patients with triple-negative breast cancer (TNBC). Preclinical data suggest that TNBC is dependent on proteasomes; however, clinical observations indicate that the efficacy of proteasome inhibitors in TNBC may be limited, suggesting the need for combination therapies. We compared bortezomib and carfilzomib and their combinations with nelfinavir and lopinavir in TNBC cell lines and primary cells with regard to their cytotoxic activity, functional proteasome inhibition, and induction of theunfolded protein response (UPR). Furthermore, we evaluated the involvement of sXBP1, ABCB1, and ABCG2 in the cytotoxic activity of drug combinations. Carfilzomib, via proteasomeβ5 + β2 inhibition, is more cytotoxic in TNBC than bortezomib, which inhibits β5 + β1 proteasome subunits. The cytotoxicity of carfilzomib was significantly potentiated by nelfinavir or lopinavir. Carfilzomib with lopinavir induced endoplasmic reticulum stress and pro-apoptotic UPR through the accumulation of excess proteasomal substrate protein in TNBC in vitro. Moreover, lopinavir increased the intracellular availability of carfilzomib by inhibiting carfilzomib export from cells that express high levels and activity of ABCB1, but not ABCG2. Proteasome inhibition by carfilzomib combined with nelfinavir/lopinavir represents a potential treatment option for TNBC, warranting further investigation.

Gastrointestinal toxicities of proteasome inhibitor therapy

PurposeProteasome inhibitors (PIs), which cause cell death via tumor suppressor and pro-apoptotic proteins, are integral to treatment of many hematologic malignancies but are limited by their gastrointestinal adverse effects. Evidence regarding these PI-related adverse effects is scant. In this study, we evaluated gastrointestinal adverse events caused by PIs and compared gastrointestinal toxicities between bortezomib, carfilzomib, and ixazomib.MethodsWe conducted a retrospective study of cancer patients treated with PIs at a tertiary care cancer center to investigate the clinical characteristics of PI-related gastrointestinal adverse events.ResultsOur sample comprised 973 patients with PI exposure and stool studies ordered between January 2017 and December 2022. Of these, 193 patients (20%) had PI-related gastrointestinal toxicity based on clinical symptoms and stool study results. The most common symptom was diarrhea, present in 169 (88% of those with gastrointestinal toxicity). Twenty-two (11%) required hospitalization, and 71 (37%) developed recurrence of symptoms. Compared to bortezomib or carfilzomib, ixazomib had a longer interval from PI initiation to the onset of gastrointestinal symptoms (313 days vs 58 days vs 89 days, p = 0.002) and a significantly lower percentage of diarrhea-predominant presentation of gastrointestinal toxicity (71% vs 96% vs 91%, p = 0.048).ConclusionWhile PI-related gastrointestinal toxicities have various presentations and courses based on different regimens, the vast majority of patients presented with milder disease behavior. Despite a considerably high rate of hospitalization and recurrence after treatment necessitating optimization of clinical management, our cohort demonstrates favorable outcomes without long-term consequences.

PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

Multiple myeloma (MM) is a Ubiquitin Proteasome System (UPS)-dysfunction disease. We previously reported that high PRAME transcript levels associated with unfavorable progression free survival (PFS) in patients with no bortezomib therapy, and bortezomib-containing regimen significantly improved PFS in patients with high PRAME transcript levels, which indicated that PRAME expression was prognostic for MM patients, and was related to proteasome inhibitor treatment. However, molecular mechanisms underlying the above clinical performance remain unclear. In the present study, MM cell models with PRAME knockdown and overexpression were established, and PRAME was identified to play the role of promoting proliferation in MM cells. P-Akt signaling was found to be activated as PRAME overexpressed. As a substrate recognizing subunit (SRS) of the E3 ubiquitin ligase, PRAME targets substrate proteins and mediates their degradation. CTMP and p21 were found to be the novel targets of PRAME in the Cul2-dependent substrate recognition process. PRAME interacted with and mediated ubiquitination and degradation of CTMP and p21, which led to accumulation of p-Akt and CCND3 proteins, and thus promoted cell proliferation and increased bortezomib sensitivity in MM cells.

RNF31-mediated IKKα ubiquitination aggravates inflammation and intestinal injury through regulating NF-κB activation in human and mouse neonates

AimsNeonatal necrotizing enterocolitis (NEC) is a leading cause of intestine inflammatory disease, and macrophage is significantly activated during NEC development. Posttranslational modifications (PTMs) of proteins, particularly ubiquitination, play critical roles in immune response. This study aimed to investigate the effects of ubiquitin-modified proteins on macrophage activation and NEC, and discover novel NEC-related inflammatory proteins. Materials and methodsProteomic and ubiquitin proteomic analyses of intestinal macrophages in NEC/healthy mouse pups were carried out. In vitro macrophage inflammation model and in vivo NEC mouse model, as well as clinical human samples were used for further verification the inhibitor of nuclear factor-κB kinase α (IKKα) ubiquitination on NEC development through Western blot, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry. Key findingsWe report here that IKKα was a new ubiquitin-modified protein during NEC through ubiquitin proteomics, and RING finger protein 31 (RNF31) acted as an E3 ligase to be involved in IKKα degradation. Inhibition of IKKα ubiquitination and degradation with siRNF31 or proteasome inhibitor decreased nuclear factor-κB (NF-κB) activation, thereby decreasing the expression of pro-inflammatory factors and M1 macrophage polarization, resulting in reliving the severity of NEC. SignificanceOur study suggests the activation of RNF31-IKKα-NF-κB axis triggering NEC development and suppressing RNF31-mediated IKKα degradation may be therapeutic strategies to be developed for NEC treatment.

O-033 Molecular insights into the decline in oocyte quality with advanced maternal age: findings from comprehensive single cell -omics analysis of 112 human oocytes

Abstract Study question How does maternal age impact the molecular composition of oocytes during their final meiotic progression? Summary answer Maternal age significantly correlates with proteomic changes during oocyte maturation, particularly in proteostasis and meiosis related proteins, with minimal impact on transcriptome and DNA-methylation patterns. What is known already Oocyte quality declines with maternal age, resulting in diminished developmental competence and higher aneuploidy rates. However, the molecular mechanisms behind this decline in oocyte quality remain elusive. Transcriptomic studies have revealed few differentially expressed genes in advanced maternal age (AMA) oocytes, suggesting that age-related changes in oocyte quality result from a complex interplay of molecular factors, rather than a single cause. However, comprehensive methylation and proteomic data are still lacking. We employed advanced -omics to assess the effect of maternal age on the molecular signature of human oocytes, focusing on DNA-methylation, transcriptome and proteome changes during the final meiotic progression. Study design, size, duration This study included a total of 112 oocytes obtained from young (&amp;lt;35 years, n = 35) and AMA women (&amp;gt;37 years, n = 55), who were recruited in the study from October 2021 to October 2023. Both germinal vesicle (GV, n = 68) and metaphase II (MII, n = 44) oocytes were analysed. Additionally, 19 immature oocytes (GV and metaphase I) from 9 young women were used for validation. Participants/materials, setting, methods Parallel single-cell bisulfite and RNA sequencing was applied to 44 oocytes (26 GV-Young, 6 MII-Young, 8 GV-AMA, 4 MII-AMA) and single-cell proteomics to 68 oocytes (18 GV-Young, 18 MII-Young, 18 GV-AMA, 14 MII-AMA). Additionally, 10 GV and 9 MI oocytes were treated with the proteasome inhibitor MG-312 (0 µM, 10 µM) for 6 hours, followed by rescue in vitro maturation (rIVM) for 36 hours. Chromosomal distribution was assessed by immunocytochemistry. Main results and the role of chance Our analysis revealed no significant changes in DNA methylation patterns in either GV nor MII oocytes associated with AMA. Also, very limited changes were detected in the transcriptome, with transcript levels of only 5 genes in GV oocytes and 7 genes in MII oocytes detected as being significantly changed. In contrast, proteomic analysis, particularly in GV oocytes, revealed significant age-related changes, notably in proteins participating in the proteostasis network (signalosome complex, UCHL1) including chaperones (TRiC-complex, HSP7C, STIP1), and in the cell cycle, including signal transduction factors (1433E, integrins) and cytoskeleton regulators (DYL2, CAPZB, ARHGG) (Rs ≤ |0.5|, p ≤ 0.05). The proteasome complex, which plays a crucial role both in meiosis and the proteostasis network, was found to decline with age; changes were evident in several subunits of the complex (e.g., PRS8, PRS6A, and PRS10; Rs ≤ -0.56, p ≤ 0.05). Compared to controls, treatment of immature oocytes with the proteasome inhibitor MG-132 resulted in either maturation failure or chromosome mislocalization in metaphase plate, further validating the essential role of the proteasome during oocyte maturation. In MII oocytes, 7 proteins showed alterations with age, including the oocyte-specific marker DDX4, which significantly declined in abundance (Rs =-0.6, p ≤ 0.05). Limitations, reasons for caution Unlike GVs that were collected fresh, MII oocytes underwent vitrification and warming before being included in the study due to clinical protocols. These procedures may have unknown effects on the transcriptome and proteome. Wider implications of the findings Our findings suggest that age primarily affects oocyte quality at the post-transcriptional level, potentially through meiosis dysregulation and proteostasis disruption. We also demonstrate the proteasome's vital role in oocyte maturation, suggesting that targeting the proteasome complex may improve oocyte quality in AMA women. Trial registration number Not applicable

A single-day polychemotherapy regimen with proteasome inhibitor combinations for relapsed/refractory myeloma in the era of novel therapies.

PCAB (prednisone, cyclophosphamide, doxorubicin, carmustine) is a single-day regimen previously used for induction and now in relapsed/refractory multiple myeloma (RRMM). We retrospectively analysed the outcomes of 85 patients from five Australian centres. These included 30 patients (35.3%) who received PCAB with one additional agent (bortezomib most frequently). Median age of the patients was 65 years (37-80), with a median of four (1-8) prior lines of therapy. ORR was 37% (CR 4.9%). Median progression free survival and overall survival were 4.4 months (95% CI 3.5-6.7) and 7.4 months (95% CI 6.4-10.2), respectively. Extramedullary disease (EMD) was associated with shorter survival. Grade 3 or 4 cytopenia and febrile neutropenia occurred in 76.2% and 39.1%, respectively, with six (7.1%) treatment-related mortalities. Median inpatient stay was 3.3 days/28-day cycle (IQR 0.6-13), and for patients who died, a median of 20.2% of days alive were spent inpatient (IQR 6.4-39.1%). Three patients were successfully bridged to CAR T-cell therapy using PCAB, despite being penta-exposed and having EMD. PCAB may be considered as a useful salvage therapy amongst other polychemotherapy regimens in late relapse. Further studies is warranted to investigate and define its role as a bridging therapy to novel therapeutics.

HRS-4642: The next piece of the puzzle to keep KRAS in check

KRASG12D is the most frequent KRAS mutation in human cancer. In this issue, Zhou etal. describe a novel KRASG12D inhibitor, HRS-4642, that shows potent and selective anti-tumor activity across various models and synergizes with proteasome inhibitors. Responses have also been observed in patients during an ongoing phase 1 trial.

High throughput screening identifies dasatinib as synergistic with trametinib in low grade serous ovarian carcinoma

BackgroundLow grade serous ovarian carcinoma (LGSOC) is a distinct histotype of ovarian cancer characterised high levels of intrinsic chemoresistance, highlighting the urgent need for new treatments. High throughput screening in clinically-informative cell-based models represents an attractive strategy for identifying candidate treatment options for prioritisation in clinical studies. MethodsWe performed a high throughput drug screen of 1610 agents across a panel of 6 LGSOC cell lines (3 RAS/RAF-mutant, 3 RAS/RAF-wildtype) to identify novel candidate therapeutic approaches. Validation comprised dose-response analysis across 9 LGSOC models and 5 high grade serous comparator lines. Results16 hits of 1610 screened compounds were prioritised for validation based on >50% reduction in nuclei counts in over half of screened cell lines at 1000 nM concentration. 11 compounds passed validation, and the four agents of greatest interest (dasatinib, tyrosine kinase inhibitor; disulfiram, aldehyde dehydrogenase inhibitor; carfilzomib, proteasome inhibitor; romidepsin, histone deacetylase inhibitor) underwent synergy profiling with the recently approved MEK inhibitor trametinib. Disulfiram demonstrated excellent selectivity for LGSOC versus high grade serous ovarian carcinoma comparator lines (P = 0.003 for IC50 comparison), while the tyrosine kinase inhibitor dasatinib demonstrated favourable synergy with trametinib across multiple LGSOC models (maximum zero interaction potency synergy score 46.9). The novel, highly selective Src family kinase (SFK) inhibitor NXP900 demonstrated a similar trametinib synergy profile to dasatinib, suggesting that SFK inhibition is the likely driver of synergy. ConclusionDasatinib and other SFK inhibitors represent novel candidate treatments for LGSOC and demonstrate synergy with trametinib. Disulfiram represents an additional treatment strategy worthy of investigation.

Elotuzumab plus pomalidomide and dexamethasone in relapsed/refractory multiple myeloma: Extended follow-up of a multicenter, retrospective real-world experience with 321 cases outside of controlled clinical trials.

The ELOQUENT-3 trial demonstrated the superiority of the combination of elotuzumab, pomalidomide, and dexamethasone (EloPd) in terms of efficacy and safety, compared to Pd in relapsed/refractory multiple myeloma (RRMM), who had received at least two prior therapies, including lenalidomide and a proteasome inhibitor. The present study is an 18-month follow-up update of a previously published Italian real-life RRMM cohort of patients treated with EloPd. This revised analysis entered 319 RRMM patients accrued in 41 Italian centers. After a median follow-up of 17.7months, 213 patients (66.4%) experienced disease progression or died. Median progression-free survival (PFS) and overall survival (OS) were 7.5 and 19.2months, respectively. The updated multivariate analysis showed a significant reduction of PFS benefit magnitude both in advanced International Staging System (ISS) (II and III) stages and previous exposure to daratumumab cases. Instead, advanced ISS (II and III) stages and more than 2 previous lines of therapy maintained an independent prognostic impact on OS. Major adverse events included grade three-fourths neutropenia (24.9%), anemia (13.4%), lymphocytopenia (15.5%), and thrombocytopenia (10.7%), while infection rates and pneumonia were 19.3% and 8.7%, respectively. A slight increase in the incidence of neutropenia and lymphocytopenia was registered with longer follow-up. In conclusion, our real-world study still confirms that EloPd is a safe and possible therapeutic choice for RRMM. Nevertheless, novel strategies are desirable for those patients exposed to daratumumab.

Nicotinamide riboside first alleviates symptoms but later downregulates dopamine metabolism in proteasome inhibition mouse model of Parkinson's disease

Parkinson's disease (PD) is associated with a reduction in 26/20S proteasome and mitochondrial function and depletion of dopamine. Activation of mitochondrial function with the NAD+ precursor nicotinamide riboside (NR) is a potential therapeutic for PD. However, despite recently started clinical trials, analysis of NR in mammalian animal PD models is lacking and data in simpler PD models is limited. We analyzed the effect of NR in C. elegans and in mouse 26/20S proteasome inhibition models of PD. In C. elegans, NR rescued α-synuclein overexpression induced phenotypes likely by activating the mitochondrial unfolded protein response. However, in a proteasome inhibitor-induced mouse model of PD, NR first partially rescued behavioural dysfunction, but later resulted in decrease in dopamine and its related gene expression in the substantia nigra. Our results suggest that reduction in 26/20S function with long term NR treatment may increase risk for developing reduced nigrostriatal DA function.