Impact of soluble BCMA and non-T-cell factors on refractoriness to BCMA-targeting T-cell engagers in multiple myeloma.

Adoptive T cell therapy is a promising therapy for multiple myeloma (MM), but its efficacy hinges on understanding relevant biological and predictive markers of response. B cell maturation antigen (BCMA) is a key target antigen in MM, with active development of multiple anti-BCMA T cell engagers (TCE) and chimeric antigen receptor T cell (CAR T) therapies. The regulation of surface BCMA expression by MM cells, resulting in the shedding of soluble BCMA (sBCMA), has triggered debate surrounding the significance of sBCMA as a predictive marker and its potential impact on treatment outcomes. In order to address this, we leveraged whole genome sequencing and in vitro assays to demonstrate that sBCMA may independently predict primary refractoriness to anti-BCMA therapies. In addition to sBCMA, tumor burden and surface BCMA antigen density collectively influence anti-BCMA TCE cytotoxic efficacy. Correlative analyses of 163 patients treated with anti-BCMA TCE teclistamab validated and further underscored the association between elevated baseline sBCMA (>400 ng/mL) and refractoriness. Importantly, increasing TCE dose, the use of TCE against alternative targets (e.g.,GPRC5D), or gamma secretase inhibitors were able to overcome high sBCMA. These findings highlight the importance of accounting for baseline sBCMA levels, disease burden, and TCE dose intensity when administering anti-BCMA TCEs, offering critical insights for optimizing therapeutic strategies to overcome specific high-risk features and primary anti-BCMA TCE refractoriness.

SARS-CoV-2 Neutralizing Antibody LY-CoV555 in Outpatients with Covid-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (Covid-19), which is most frequently mild yet can be severe and life-threatening. Virus-neutralizing monoclonal antibodies are predicted to reduce viral load, ameliorate symptoms, and prevent hospitalization. METHODS: In this ongoing phase 2 trial involving outpatients with recently diagnosed mild or moderate Covid-19, we randomly assigned 452 patients to receive a single intravenous infusion of neutralizing antibody LY-CoV555 in one of three doses (700 mg, 2800 mg, or 7000 mg) or placebo and evaluated the quantitative virologic end points and clinical outcomes. The primary outcome was the change from baseline in the viral load at day 11. The results of a preplanned interim analysis as of September 5, 2020, are reported here. RESULTS: At the time of the interim analysis, the observed mean decrease from baseline in the log viral load for the entire population was -3.81, for an elimination of more than 99.97% of viral RNA. For patients who received the 2800-mg dose of LY-CoV555, the difference from placebo in the decrease from baseline was -0.53 (95% confidence interval [CI], -0.98 to -0.08; P = 0.02), for a viral load that was lower by a factor of 3.4. Smaller differences from placebo in the change from baseline were observed among the patients who received the 700-mg dose (-0.20; 95% CI, -0.66 to 0.25; P = 0.38) or the 7000-mg dose (0.09; 95% CI, -0.37 to 0.55; P = 0.70). On days 2 to 6, the patients who received LY-CoV555 had a slightly lower severity of symptoms than those who received placebo. The percentage of patients who had a Covid-19-related hospitalization or visit to an emergency department was 1.6% in the LY-CoV555 group and 6.3% in the placebo group. CONCLUSIONS: In this interim analysis of a phase 2 trial, one of three doses of neutralizing antibody LY-CoV555 appeared to accelerate the natural decline in viral load over time, whereas the other doses had not by day 11. (Funded by Eli Lilly; BLAZE-1 ClinicalTrials.gov number, NCT04427501.).

Bamlanivimab plus Etesevimab in Mild or Moderate Covid-19.

BACKGROUND: Patients with underlying medical conditions are at increased risk for severe coronavirus disease 2019 (Covid-19). Whereas vaccine-derived immunity develops over time, neutralizing monoclonal-antibody treatment provides immediate, passive immunity and may limit disease progression and complications. METHODS: In this phase 3 trial, we randomly assigned, in a 1:1 ratio, a cohort of ambulatory patients with mild or moderate Covid-19 who were at high risk for progression to severe disease to receive a single intravenous infusion of either a neutralizing monoclonal-antibody combination agent (2800 mg of bamlanivimab and 2800 mg of etesevimab, administered together) or placebo within 3 days after a laboratory diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The primary outcome was the overall clinical status of the patients, defined as Covid-19-related hospitalization or death from any cause by day 29. RESULTS: A total of 1035 patients underwent randomization and received an infusion of bamlanivimab-etesevimab or placebo. The mean (±SD) age of the patients was 53.8±16.8 years, and 52.0% were adolescent girls or women. By day 29, a total of 11 of 518 patients (2.1%) in the bamlanivimab-etesevimab group had a Covid-19-related hospitalization or death from any cause, as compared with 36 of 517 patients (7.0%) in the placebo group (absolute risk difference, -4.8 percentage points; 95% confidence interval [CI], -7.4 to -2.3; relative risk difference, 70%; P<0.001). No deaths occurred in the bamlanivimab-etesevimab group; in the placebo group, 10 deaths occurred, 9 of which were designated by the trial investigators as Covid-19-related. At day 7, a greater reduction from baseline in the log viral load was observed among patients who received bamlanivimab plus etesevimab than among those who received placebo (difference from placebo in the change from baseline, -1.20; 95% CI, -1.46 to -0.94; P<0.001). CONCLUSIONS: Among high-risk ambulatory patients, bamlanivimab plus etesevimab led to a lower incidence of Covid-19-related hospitalization and death than did placebo and accelerated the decline in the SARS-CoV-2 viral load. (Funded by Eli Lilly; BLAZE-1 ClinicalTrials.gov number, NCT04427501.).

Efficacy and Safety of Continued Treatment With Mirikizumab in a Phase 2 Trial of Patients With Ulcerative Colitis.

BACKGROUND & AIMS: Mirikizumab is an antibody against the p19 subunit of interleukin 23 that has demonstrated clinical efficacy and was well tolerated following 12 weeks of induction treatment in a phase 2 trial of patients with moderate to severe ulcerative colitis. We present results of the open-label extended induction period in patients who did not initially respond to treatment with mirikizumab. METHODS: This study was a continuation of I6T-MC-AMAC, a double-blind trial, performed at 75 sites in 14 countries, in which patients with moderate to severe ulcerative colitis were randomly assigned to 12 weeks induction therapy with 50 mg, 200 mg, or 600 mg mirikizumab or placebo. Patients without a clinical response (a 9-point decrease in Mayo subscore of ≥2 points and ≥35% from baseline and either a decrease of rectal bleeding subscore of ≥1 or a rectal bleeding subscore of 0 or 1) at week 12 were offered the opportunity to participate in an open-label, extended induction study for another 12 weeks, in which they received either 600 mg intravenous mirikizumab (n = 20) or, following a protocol amendment, 1000 mg intravenous mirikizumab (n = 64) every 4 weeks. At week 24, patients with a clinical response continued the extension maintenance period and received 200 mg subcutaneous mirikizumab. Endpoints included clinical remission (Mayo subscores of 0 for rectal bleeding, 0 or 1 with a 1-point decrease from baseline), clinical response, endoscopic remission (Mayo endoscopic subscore of 0), or endoscopic improvement (endoscopic subscore of 0 or 1), at study weeks 24 and 52. Data were analysed for patients who received mirikizumab or placebo during the induction phase of the study. RESULTS: Among participants who did not respond to induction mirikizumab, 50.0% of those who received the 12-week extension of 600 mg mirikizumab and 43.8% who received the extension of 1000 mg mirikizumab achieved a clinical response; 15.0% and 9.4% achieved clinical remission, respectively. Endoscopic improvement was achieved by 20.0% of subjects in the 600 mg mirikizumab group and 15.6% subjects in the 1000 mg mirikizumab group. Among initial nonresponders to mirikizumab who had clinical response at study week 24 and continued into maintenance therapy, 65.8% maintained the clinical response, 26.3% achieved clinical remission, and 34.2% had endoscopic improvement at week 52. No new safety concerns were identified. CONCLUSIONS: Extended doses of mirikizumab (600 mg and 1000 mg) for an additional 12 weeks produce a clinical response in up to 50% of patients who did not have a clinical response to 12 weeks of induction doses (50 mg, 200 mg, or 600 mg). Most of the responders to the extended doses maintained clinical response for up to 52 weeks. Clinicaltrials.gov no: NCT02589665.

Uphyloplot2: visualizing phylogenetic trees from single-cell RNA-seq data.

BACKGROUND: Recent advances in single cell sequencing technologies allow for greater resolution in assessing tumor clonality using chromosome copy number variations (CNVs). While single cell DNA sequencing technologies are ideal to identify tumor sub-clones, they remain expensive and in contrast to single cell RNA-seq (scRNA-seq) methods are more limited in the data they generate. However, CNV data can be inferred from scRNA-seq and bulk RNA-seq, for which several tools have been developed, including inferCNV, CaSpER, and HoneyBADGER. Inferences regarding tumor clonality from CNV data (and other sources) are frequently visualized using phylogenetic plots, which previously required time-consuming and error-prone, manual analysis. RESULTS: Here, we present Uphyloplot2, a python script that generates phylogenetic plots directly from inferred RNA-seq data, or any Newick formatted dendrogram file. The tool is publicly available at https://github.com/harbourlab/UPhyloplot2/ . CONCLUSIONS: Uphyloplot2 is an easy-to-use tool to generate phylogenetic plots to depict tumor clonality from scRNA-seq data and other sources.

Efficacy and Safety of Mirikizumab in a Randomized Phase 2 Study of Patients With Ulcerative Colitis.

BACKGROUND & AIMS: Interleukin 23 contributes to the pathogenesis of ulcerative colitis (UC). We investigated the effects of mirikizumab, a monoclonal antibody against the p19 subunit of interleukin 23, in a phase 2 study of patients with UC. METHODS: We performed a trial of the efficacy and safety of mirikizumab in patients with moderate to severely active UC, enrolling patients from 14 countries from January 2016 through September 2017. Patients were randomly assigned to groups given intravenous placebo (N = 63), mirikizumab 50 mg (N = 63) or 200 mg (N = 62) with exposure-based dosing, or mirikizumab 600 mg with fixed dosing (N = 61) at weeks 0, 4, and 8. Of assigned patients, 63% had prior exposure to a biologic agent. Clinical responders (decrease in 9-point Mayo score, including ≥2 points and ≥35% from baseline with either a decrease of rectal bleeding subscore of ≥1 or a rectal bleeding subscore of 0 or 1) at week 12 who had received mirikizumab were randomly assigned to groups that received maintenance treatment with mirikizumab 200 mg subcutaneously every 4 weeks (N = 47) or every 12 weeks (N = 46). The primary endpoint was clinical remission (Mayo subscores of 0 for rectal bleeding, with 1-point decrease from baseline for stool frequency, and 0 or 1 for endoscopy) at week 12. A multiple testing procedure was used that began with the 600-mg dose group, and any nonsignificant comparison result ended the formal statistical testing procedure. RESULTS: At week 12, 15.9% (P = .066), 22.6% (P = .004), and 11.5% (P = .142) of patients in the 50-mg, 200-mg, and 600-mg groups achieved clinical remission, respectively, compared with 4.8% of patients given placebo. The primary endpoint was not significant (comparison to 600 mg, P > .05). Clinical responses occurred in 41.3% (P = .014), 59.7% (P < .001), and 49.2% (P = .001) of patients in the 50-mg, 200-mg, and 600-mg groups, respectively, compared with 20.6% of patients given placebo. At week 52, 46.8% of patients given subcutaneous mirikizumab 200 mg every 4 weeks and 37.0% given subcutaneous mirikizumab 200 mg every 12 weeks were in clinical remission. CONCLUSIONS: In a randomized trial of patients with UC, mirikizumab was effective in inducing a clinical response after 12 weeks. Additional studies are required to determine the optimal dose for induction of remission. Mirikizumab showed durable efficacy throughout the maintenance period. Clinicaltrials.gov, Number NCT02589665.

Effect of Bamlanivimab as Monotherapy or in Combination With Etesevimab on Viral Load in Patients With Mild to Moderate COVID-19: A Randomized Clinical Trial.

Importance: Coronavirus disease 2019 (COVID-19) continues to spread rapidly worldwide. Neutralizing antibodies are a potential treatment for COVID-19. Objective: To determine the effect of bamlanivimab monotherapy and combination therapy with bamlanivimab and etesevimab on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in mild to moderate COVID-19. Design, Setting, and Participants: The BLAZE-1 study is a randomized phase 2/3 trial at 49 US centers including ambulatory patients (N = 613) who tested positive for SARS-CoV-2 infection and had 1 or more mild to moderate symptoms. Patients who received bamlanivimab monotherapy or placebo were enrolled first (June 17-August 21, 2020) followed by patients who received bamlanivimab and etesevimab or placebo (August 22-September 3). These are the final analyses and represent findings through October 6, 2020. Interventions: Patients were randomized to receive a single infusion of bamlanivimab (700 mg [n = 101], 2800 mg [n = 107], or 7000 mg [n = 101]), the combination treatment (2800 mg of bamlanivimab and 2800 mg of etesevimab [n = 112]), or placebo (n = 156). Main Outcomes and Measures: The primary end point was change in SARS-CoV-2 log viral load at day 11 (±4 days). Nine prespecified secondary outcome measures were evaluated with comparisons between each treatment group and placebo, and included 3 other measures of viral load, 5 on symptoms, and 1 measure of clinical outcome (the proportion of patients with a COVID-19-related hospitalization, an emergency department [ED] visit, or death at day 29). Results: Among the 577 patients who were randomized and received an infusion (mean age, 44.7 [SD, 15.7] years; 315 [54.6%] women), 533 (92.4%) completed the efficacy evaluation period (day 29). The change in log viral load from baseline at day 11 was -3.72 for 700 mg, -4.08 for 2800 mg, -3.49 for 7000 mg, -4.37 for combination treatment, and -3.80 for placebo. Compared with placebo, the differences in the change in log viral load at day 11 were 0.09 (95% CI, -0.35 to 0.52; P = .69) for 700 mg, -0.27 (95% CI, -0.71 to 0.16; P = .21) for 2800 mg, 0.31 (95% CI, -0.13 to 0.76; P = .16) for 7000 mg, and -0.57 (95% CI, -1.00 to -0.14; P = .01) for combination treatment. Among the secondary outcome measures, differences between each treatment group vs the placebo group were statistically significant for 10 of 84 end points. The proportion of patients with COVID-19-related hospitalizations or ED visits was 5.8% (9 events) for placebo, 1.0% (1 event) for 700 mg, 1.9% (2 events) for 2800 mg, 2.0% (2 events) for 7000 mg, and 0.9% (1 event) for combination treatment. Immediate hypersensitivity reactions were reported in 9 patients (6 bamlanivimab, 2 combination treatment, and 1 placebo). No deaths occurred during the study treatment. Conclusions and Relevance: Among nonhospitalized patients with mild to moderate COVID-19 illness, treatment with bamlanivimab and etesevimab, compared with placebo, was associated with a statistically significant reduction in SARS-CoV-2 viral load at day 11; no significant difference in viral load reduction was observed for bamlanivimab monotherapy. Further ongoing clinical trials will focus on assessing the clinical benefit of antispike neutralizing antibodies in patients with COVID-19 as a primary end point. Trial Registration: ClinicalTrials.gov Identifier: NCT04427501.

Gain of function of ASXL1 truncating protein in the pathogenesis of myeloid malignancies.

Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic mouse model, Asxl1Y588X Tg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.

The immediate effects of knee flexion range of motion following manual therapy or self-stretching/active range of motion following a total knee arthroplasty: a case report.

[Purpose] The purposes of this case study are to: (1) report the immediate effects of knee flexion range of motion following manual therapy (MT) and self-stretching/AROM following a total knee arthroplasty (TKA). (2) contribute empirical evidence to the literature through reports within this case study. [Participant and Methods] For 6 days, the authors utilized a different MT technique and 1 day of self-stretching and active range of motion for an 85-year-old male who was 3 days status post right TKA. [Results] The greatest gains for AROM/PROM for knee flexion were achieved while performing typical arthrokinematic motion joint mobilizations, for AROM and PROM, resulting in a gain of 10 degrees and 10 degrees, respectfully. [Conclusion] We theorize that performing typical arthrokinematic motion joint mobilizations stimulates a greater response from the mechanoreceptors and therefore a greater stimulation response to the central and peripheral nervous systems. This greater stimulation may explain the greatest immediate gain in knee flexion range of motion being performed by typical arthrokinematic motion joint mobilizations. The outcomes of this study demonstrate the start of some empirical evidence while exploring the immediate effects of knee flexion range of motion following manual therapy and self-stretching/AROM following a TKA.

Timing antigenic escape in multiple myeloma treated with T-cell redirecting immunotherapies.

Recent data highlight genomic events driving antigen escape as a recurring cause of chimeric antigen receptor T-cell (CAR-T) and bispecific T-cell engager (TCE) resistance in multiple myeloma (MM). Yet, it remains unclear if these events, leading to clonal dominance at progression, result from acquisition under treatment selection or selection of pre-existing undetectable clones. This differentiation gains importance as these immunotherapies progress to earlier lines of treatment, prompting the need for innovative diagnostic testing to detect these events early on. By reconstructing phylogenetic trees and exploring chemotherapy mutational signatures as temporal barcodes in 11 relapsed refractory MM patients with available whole genome sequencing data before and after CART/TCE treatment, we demonstrated that somatic antigen escape mechanisms for BCMA- and GPRC5D-targeting therapies are acquired post-diagnosis, likely during CART/TCE treatment. Longitudinal tracking of these mutations using digital PCR in 4 patients consistently showed that genomic events promoting antigen escape were not detectable during the initial months of therapy but began to emerge nearly 1 year post therapy initiation. This finding reduces the necessity for a diagnostic panel to identify these events before CART/TCE. Instead, it underscores the importance of surveillance and identifying patients at higher risk of acquiring these events.

Genomic Profiling to Contextualize the Results of Intervention for Smoldering Multiple Myeloma.

PURPOSE: Early intervention for high-risk smoldering multiple myeloma (HR-SMM) achieves deep and prolonged responses. It is unclear if beneficial outcomes are due to the treatment of less complex, susceptible disease or inaccuracy in clinical definition of cases entered. EXPERIMENTAL DESIGN: In this study, we interrogated whole-genome and whole-exome sequencing for 54 patients across two HR-SMM interventional studies (NCT01572480 and NCT02279394). RESULTS: We reveal that the genomic landscape of treated HR-SMM is generally simple as compared with newly diagnosed multiple myeloma counterparts with less inactivation of tumor suppressor genes, RAS pathway mutations, MYC disruption, and APOBEC contribution. The absence of these events parallels that of indolent precursor conditions, possibly explaining overall excellent outcomes. However, some patients harboring genomic complexity fail to sustain response and experience resistant, progressive disease. Overall, clinical risk scores do not effectively discriminate between genomically indolent and aggressive disease. CONCLUSIONS: Genomic profiling can contextualize the advantage of early intervention in SMM and guide personalization of therapy. See related commentary by Weinhold and Rasche, p. 4263.

PRAME induces genomic instability in uveal melanoma.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME that can be targeted therapeutically in cancer.

Genomic Classification and Individualized Prognosis in Multiple Myeloma.

PURPOSE: Outcomes for patients with newly diagnosed multiple myeloma (NDMM) are heterogenous, with overall survival (OS) ranging from months to over 10 years. METHODS: To decipher and predict the molecular and clinical heterogeneity of NDMM, we assembled a series of 1,933 patients with available clinical, genomic, and therapeutic data. RESULTS: Leveraging a comprehensive catalog of genomic drivers, we identified 12 groups, expanding on previous gene expression-based molecular classifications. To build a model predicting individualized risk in NDMM (IRMMa), we integrated clinical, genomic, and treatment variables. To correct for time-dependent variables, including high-dose melphalan followed by autologous stem-cell transplantation (HDM-ASCT), and maintenance therapy, a multi-state model was designed. The IRMMa model accuracy was significantly higher than all comparator prognostic models, with a c-index for OS of 0.726, compared with International Staging System (ISS; 0.61), revised-ISS (0.572), and R2-ISS (0.625). Integral to model accuracy was 20 genomic features, including 1q21 gain/amp, del 1p, TP53 loss, NSD2 translocations, APOBEC mutational signatures, and copy-number signatures (reflecting the complex structural variant chromothripsis). IRMMa accuracy and superiority compared with other prognostic models were validated on 256 patients enrolled in the GMMG-HD6 (ClinicalTrials.gov identifier: NCT02495922) clinical trial. Individualized patient risks were significantly affected across the 12 genomic groups by different treatment strategies (ie, treatment variance), which was used to identify patients for whom HDM-ASCT is particularly effective versus patients for whom the impact is limited. CONCLUSION: Integrating clinical, demographic, genomic, and therapeutic data, to our knowledge, we have developed the first individualized risk-prediction model enabling personally tailored therapeutic decisions for patients with NDMM.

Conversion strategy using an expanded genetic alphabet to assay nucleic acids.

Methods to detect DNA and RNA (collectively xNA) are easily plagued by noise, false positives, and false negatives, especially with increasing levels of multiplexing in complex assay mixtures. Here, we describe assay architectures that mitigate these problems by converting standard xNA analyte sequences into sequences that incorporate nonstandard nucleotides (Z and P). Z and P are extra DNA building blocks that form tight nonstandard base pairs without cross-binding to natural oligonucleotides containing G, A, C, and T (GACT). The resulting improvements are assessed in an assay that inverts the standard Luminex xTAG architecture, placing a biotin on a primer (rather than on a triphosphate). This primer is extended on the target to create a standard GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of competition from any natural oligonucleotides, even in complex biological samples.

A randomized and open-label trial evaluating the addition of pazopanib to lapatinib as first-line therapy in patients with HER2-positive advanced breast cancer.

This phase II study (VEG20007; NCT00347919) with randomized and open-label components evaluated first-line lapatinib plus pazopanib therapy and/or lapatinib monotherapy in patients with human epidermal growth factor receptor type 2 (HER2)-positive advanced/metastatic breast cancer. Patients were enrolled sequentially into two cohorts: Cohort 1, patients were randomly assigned to lapatinib 1,000 mg plus pazopanib 400 mg or lapatinib 1,500 mg monotherapy; Cohort 2, patients received lapatinib 1,500 mg plus pazopanib 800 mg. The primary endpoint was week-12 progressive disease rate (PDR) for Cohort 1. The principal secondary endpoint was week-12 response rate (RR) for Cohort 2. Efficacy was assessed in patients with centrally confirmed HER2 positivity (modified intent-to-treat population [MITT]). The study enrolled 190 patients (Cohort 1, combination n = 77, lapatinib n = 73; Cohort 2, n = 40). The MITT population comprised n = 141 (Cohort 1) and n = 36 (Cohort 2). In Cohort 1, week-12 PDRs were 36.2 % (combination) versus 38.9 % (lapatinib; P = 0.37 for the difference). Week-12 RRs were 36.2 % (combination) versus 22.2 % (lapatinib). In Cohort 2, week-12 RR was 33.3 %. In Cohort 1, grade 3/4 adverse events (AEs) included diarrhea (combination, 9 %; lapatinib, 5 %) and hypertension (combination, 5 %; lapatinib, 0 %). Grades 3/4 AEs in Cohort 2 included diarrhea (40 %), hypertension (5 %), and fatigue (5 %). Alanine aminotransferase elevations >5 times the upper limit of normal occurred in Cohort 1 (combination, 18 %; lapatinib, 5 %) and Cohort 2 (20 %). Upon conclusion, the combination of lapatinib plus pazopanib did not improve PDR compared with lapatinib monotherapy, although RR was increased. Toxicity was higher with the combination, including increased diarrhea and liver enzyme elevations.

PRAME induces genomic instability in uveal melanoma.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME than can be targeted therapeutically in cancer.

Mechanisms of antigen escape from BCMA- or GPRC5D-targeted immunotherapies in multiple myeloma.

B cell maturation antigen (BCMA) target loss is considered to be a rare event that mediates multiple myeloma (MM) resistance to anti-BCMA chimeric antigen receptor T cell (CAR T) or bispecific T cell engager (TCE) therapies. Emerging data report that downregulation of G-protein-coupled receptor family C group 5 member D (GPRC5D) protein often occurs at relapse after anti-GPRC5D CAR T therapy. To examine the tumor-intrinsic factors that promote MM antigen escape, we performed combined bulk and single-cell whole-genome sequencing and copy number variation analysis of 30 patients treated with anti-BCMA and/or anti-GPRC5D CAR T/TCE therapy. In two cases, MM relapse post-TCE/CAR T therapy was driven by BCMA-negative clones harboring focal biallelic deletions at the TNFRSF17 locus at relapse or by selective expansion of pre-existing subclones with biallelic TNFRSF17 loss. In another five cases of relapse, newly detected, nontruncating, missense mutations or in-frame deletions in the extracellular domain of BCMA negated the efficacies of anti-BCMA TCE therapies, despite detectable surface BCMA protein expression. In the present study, we also report four cases of MM relapse with biallelic mutations of GPRC5D after anti-GPRC5D TCE therapy, including two cases with convergent evolution where multiple subclones lost GPRC5D through somatic events. Immunoselection of BCMA- or GPRC5D-negative or mutant clones is an important tumor-intrinsic driver of relapse post-targeted therapies. Mutational events on BCMA confer distinct sensitivities toward different anti-BCMA therapies, underscoring the importance of considering the tumor antigen landscape for optimal design and selection of targeted immunotherapies in MM.

RB1 loss triggers dependence on ESRRG in retinoblastoma.

Retinoblastoma (Rb) is a deadly childhood eye cancer that is classically initiated by inactivation of the RB1 tumor suppressor. Clinical management continues to rely on nonspecific chemotherapeutic agents that are associated with treatment resistance and toxicity. Here, we analyzed 103 whole exomes, 20 whole transcriptomes, 5 single-cell transcriptomes, and 4 whole genomes from primary Rb tumors to identify previously unknown Rb dependencies. Several recurrent genomic aberrations implicate estrogen-related receptor gamma (ESRRG) in Rb pathogenesis. RB1 directly interacts with and inhibits ESRRG, and RB1 loss uncouples ESRRG from negative regulation. ESRRG regulates genes involved in retinogenesis and oxygen metabolism in Rb cells. ESRRG is preferentially expressed in hypoxic Rb cells in vivo. Depletion or inhibition of ESRRG causes marked Rb cell death, which is exacerbated in hypoxia. These findings reveal a previously unidentified dependency of Rb cells on ESRRG, and they implicate ESRRG as a potential therapeutic vulnerability in Rb.

HDAC11 activity contributes to MEK inhibitor escape in uveal melanoma.

We previously demonstrated that pan-HDAC inhibitors could limit escape from MEK inhibitor (MEKi) therapy in uveal melanoma (UM) through suppression of AKT and YAP/TAZ signaling. Here, we focused on the role of specific HDACs in therapy adaptation. Class 2 UM displayed higher expression of HDACs 1, 2, and 3 than Class 1, whereas HDACs 6, 8, and 11 were uniformly expressed. Treatment of UM cells with MEKi led to modulation of multiple HDACs, with the strongest increases observed in HDAC11. RNA-seq analysis showed MEKi to decrease the expression of multiple HDAC11 target genes. Silencing of HDAC11 significantly reduced protein deacetylation, enhanced the apoptotic response to MEKi and reduced growth in long-term colony formation assays across multiple UM cell lines. Knockdown of HDAC11 led to decreased expression of TAZ in some UM cell lines, accompanied by decreased YAP/TAZ transcriptional activity and reduced expression of multiple YAP/TAZ target genes. Further studies showed this decrease in TAZ expression to be associated with increased LKB1 activation and modulation of glycolysis. In an in vivo model of uveal melanoma, silencing of HDAC11 limited the escape to MEKi therapy, an effect associated with reduced levels of Ki67 staining and increased cleaved caspase-3. We have demonstrated a novel role for adaptive HDAC11 activity in UM cells, that in some cases modulates YAP/TAZ signaling leading to MEKi escape.

PieParty: visualizing cells from scRNA-seq data as pie charts.

Single-cell RNA sequencing (scRNA-seq) has been a transformative technology in many research fields. Dimensional reduction techniques such as UMAP and tSNE are used to visualize scRNA-seq data in two or three dimensions for cells to be clustered in biologically meaningful ways. Subsequently, gene expression is frequently mapped onto these plots to show the distribution of gene expression across the plots, for instance to distinguish cell types. However, plotting each cell with only a single color leads to repetitive and unintuitive representations. Here, we present PieParty, which allows scRNA-seq data to be plotted such that every cell is represented as a pie chart, and every slice in the pie charts corresponds to the gene expression of a single gene. This allows for the simultaneous visualization of the expression of multiple genes and gene networks. The resulting figures are information dense, space efficient, and highly intuitive. PieParty is publicly available on GitHub at https://github.com/harbourlab/PieParty.