A phase I study of selinexor combined with weekly carfilzomib and dexamethasone in relapsed/refractory multiple myeloma.

We performed a phase I study of weekly selinexor, carfilzomib, and dexamethasone (wSKd) in patients with relapsed/refractory multiple myeloma (MM). The primary objective was to identify the maximum tolerated dose (MTD) of wSKd. Secondary endpoints included overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Prior exposure/refractoriness to carfilzomib was permitted. Thirty patients were enrolled; 26 (87%) had triple-class exposed disease and 6 (20%) received chimeric antigen receptor (CAR) T-cell therapy. Dose level 2 (carfilzomib 70 mg/m2 Intravenous [IV] on Days 1, 8, and 15; selinexor 100 mg PO on Days 1, 8, 15, 22; dexamethasone 40 mg on Days 1, 8, 15, 22 of 28-day cycles) was chosen as the MTD, with no DLTs having occurred. The most common hematologic adverse events (AE) were thrombocytopenia (83%), anemia (70%), lymphopenia (50%), and neutropenia (50%). The most common nonhematologic AE were fatigue (70%), nausea (70%), diarrhea (53%), and anorexia (47%). The ORR was 21/30 (70%) overall and 18/23 (78%) at the MTD. At a median follow-up of 12.3 months, the median PFS was 5.3 months and median OS 23.3 months. Responses were similar in carfilzomib naïve and exposed patients. Long-term efficacy of wSKd is modest; wSKd could be employed as a bridging strategy to immunotherapies.

Phase 1/2 study of carfilzomib, pomalidomide, and dexamethasone with and without daratumumab in relapsed multiple myeloma.

We conducted a phase 1/2 study of carfilzomib, pomalidomide, and dexamethasone (KPd) and KPd with daratumumab (Dara-KPd) in relapsed/refractory multiple myeloma. The primary end points were identification of a maximum tolerated dose (MTD) of KPd for phase 1, and rates of overall response (ORR) and near complete response (nCR) after 4 cycles of KPd and Dara-KPd, respectively, for phase 2. The MTD for KPd was carfilzomib 20/27 mg/m2 on days 1, 2, 8, 9, 15, and 16 (cycles 1-8) and days 1, 2, 15, and 16 for cycles 9 and beyond; oral pomalidomide 4 mg on days 1 to 21; and oral dexamethasone 40 mg weekly in 28-day cycles. Sixty-six patients received KPd, including 34 at the MTD. The ORR after 4 cycles of KPd at the MTD was 27/34 (79%; 95% confidence interval [CI], 62%-91%), meeting the statistical threshold for efficacy. At a median follow-up of 44 months, the median progression-free survival (PFS) was 13 months and overall survival (OS) 44 months. Twenty-eight patients received Dara-KPd. The rate of nCR or better after 4 cycles was 11/28 (39%; 95% CI, 22%-59%), meeting the statistical threshold for efficacy. As the best response to Dara-KPd, the ORR was 25/28 (89%) and the rate of measurable residual disease negativity by flow cytometry (10-5) was 17/26 (65%). At a median follow-up of 26 months, the median PFS and OS for Dara-KPd were not reached. Dara-KPd induced deeper and more durable responses than KPd without compromising safety in a predominantly high-risk, lenalidomide-refractory population, warranting further evaluation of this quadruplet. This trial is registered at www.clinicaltrials.gov as #NCT01665794.

Elotuzumab and Weekly Carfilzomib, Lenalidomide, and Dexamethasone in Patients With Newly Diagnosed Multiple Myeloma Without Transplant Intent: A Phase 2 Measurable Residual Disease-Adapted Study.

Importance: Treatment of newly diagnosed multiple myeloma (NDMM) with a quadruplet regimen consisting of a monoclonal antibody, proteasome inhibitor, immunomodulatory imide, and corticosteroid has been associated with improved progression-free survival (PFS) compared with triplet regimens. The optimal quadruplet combination, and whether this obviates the need for frontline autologous stem cell transplant (ASCT), remains unknown. We evaluated elotuzumab and weekly carfilzomib, lenalidomide, and dexamethasone (Elo-KRd) without ASCT in NDMM. Objective: To investigate the efficacy of Elo-KRd using a measurable residual disease (MRD)-adapted design in NDMM regardless of ASCT eligibility. Design, Setting, and Participants: This multicenter, single-arm, phase 2 study enrolled patients between July 2017 and February 2021. Median follow-up was 29 months. Interventions: Twelve to 24 cycles of Elo-KRd; consecutive MRD-negative results at 10-6 by next-generation sequencing (NGS) after cycles 8 (C8) and 12 determined the duration of Elo-KRd. This was followed by Elo-Rd (no carfilzomib) maintenance therapy until disease progression. Main Outcomes and Measures: The primary end point was the rate of stringent complete response (sCR) and/or MRD-negativity (10-5) after C8 Elo-KRd. Secondary end points included safety, rate of response, MRD status, PFS, and overall survival (OS). As an exploratory analysis, MRD was assessed using liquid chromatography mass spectrometry (MS) on peripheral blood samples. Results: Forty-six patients were enrolled (median age 62 years, 11 [24%] aged >70 years). Overall, 32 (70%) were White, 6 (13%) were Black, 3 (6%) were more than 1 race, and 5 (11%) were of unknown race. Thirty-three (72%) were men and 13 (28%) were women. High-risk cytogenetic abnormalities were present in 22 (48%) patients. The rate of sCR and/or MRD-negativity after C8 was 26 of 45 (58%), meeting the predefined statistical threshold for efficacy. Responses deepened over time, with the MRD-negativity (10-5) rate increasing to 70% and MS-negativity rate increasing to 65%; concordance between MRD by NGS and MS increased over time. The most common (>10%) grade 3 or 4 adverse events were lung and nonpulmonary infections (13% and 11%, respectively). There was 1 grade 5 myocardial infarction. The estimated 3-year PFS was 72% overall and 92% for patients with MRD-negativity (10-5) at C8. Conclusions and Relevance: An MRD-adapted design using elotuzumab and weekly KRd without ASCT showed a high rate of sCR and/or MRD-negativity and durable responses. This approach provides support for further evaluation of MRD-guided deescalation of therapy to decrease treatment exposure while sustaining deep responses. Trial Registration: ClinicalTrials.gov Identifier: NCT02969837.

Carfilzomib, lenalidomide, and dexamethasone plus transplant in newly diagnosed multiple myeloma.

In this phase 2 multicenter study, we evaluated the incorporation of autologous stem cell transplantation (ASCT) into a carfilzomib-lenalidomide-dexamethasone (KRd) regimen for patients with newly diagnosed multiple myeloma (NDMM). Transplant-eligible patients with NDMM received 4 cycles of KRd induction, ASCT, 4 cycles of KRd consolidation, and 10 cycles of KRd maintenance. The primary end point was rate of stringent complete response (sCR) after 8 cycles of KRd with a predefined threshold of ≥50% to support further study. Seventy-six patients were enrolled with a median age of 59 years (range, 40-76 years), and 35.5% had high-risk cytogenetics. The primary end point was met, with an sCR rate of 60% after 8 cycles. Depth of response improved over time. On intent-to-treat (ITT), the sCR rate reached 76%. The rate of minimal residual disease (MRD) negativity using modified ITT was 70% according to next-generation sequencing (<10-5 sensitivity). After median follow-up of 56 months, 5-year progression-free survival (PFS) and overall survival (OS) rates were 72% and 84% for ITT, 85% and 91% for MRD-negative patients, and 57% and 72% for patients with high-risk cytogenetics. For high-risk patients who were MRD negative, 5-year rates were 77% and 81%. Grade 3 to 4 adverse events included neutropenia (34%), lymphopenia (32%), infection (22%), and cardiac events (3%). There was no grade 3 to 4 peripheral neuropathy. Patients with NDMM treated with KRd with ASCT achieved high rates of sCR and MRD-negative disease at the end of KRd consolidation. Extended KRd maintenance after consolidation contributed to deepening of responses and likely to prolonged PFS and OS. Safety and tolerability were manageable. This trial was registered at www.clinicaltrials.gov as #NCT01816971.

Potent anti-myeloma activity of the TOPK inhibitor OTS514 in pre-clinical models.

Multiple myeloma (MM) continues to be considered incurable, necessitating new drug discovery. The mitotic kinase T-LAK cell-originated protein kinase/PDZ-binding kinase (TOPK/PBK) is associated with proliferation of tumor cells, maintenance of cancer stem cells, and poor patient prognosis in many cancers. In this report, we demonstrate potent anti-myeloma effects of the TOPK inhibitor OTS514 for the first time. OTS514 induces cell cycle arrest and apoptosis at nanomolar concentrations in a series of human myeloma cell lines (HMCL) and prevents outgrowth of a putative CD138+ stem cell population from MM patient-derived peripheral blood mononuclear cells. In bone marrow cells from MM patients, OTS514 treatment exhibited preferential killing of the malignant CD138+ plasma cells compared with the CD138- compartment. In an aggressive mouse xenograft model, OTS964 given orally at 100 mg/kg 5 days per week was well tolerated and reduced tumor size by 48%-81% compared to control depending on the initial graft size. FOXO3 and its transcriptional targets CDKN1A (p21) and CDKN1B (p27) were elevated and apoptosis was induced with OTS514 treatment of HMCLs. TOPK inhibition also induced loss of FOXM1 and disrupted AKT, p38 MAPK, and NF-κB signaling. The effects of OTS514 were independent of p53 mutation or deletion status. Combination treatment of HMCLs with OTS514 and lenalidomide produced synergistic effects, providing a rationale for the evaluation of TOPK inhibition in existing myeloma treatment regimens.

Neonatal acquisition of Clostridia species protects against colonization by bacterial pathogens.

The high susceptibility of neonates to infections has been assumed to be due to immaturity of the immune system, but the mechanism remains unclear. By colonizing adult germ-free mice with the cecal contents of neonatal and adult mice, we show that the neonatal microbiota is unable to prevent colonization by two bacterial pathogens that cause mortality in neonates. The lack of colonization resistance occurred when Clostridiales were absent in the neonatal microbiota. Administration of Clostridiales, but not Bacteroidales, protected neonatal mice from pathogen infection and abrogated intestinal pathology upon pathogen challenge. Depletion of Clostridiales also abolished colonization resistance in adult mice. The neonatal bacteria enhanced the ability of protective Clostridiales to colonize the gut.

The composition of the microbiota modulates allograft rejection.

Transplantation is the only cure for end-stage organ failure, but without immunosuppression, T cells rapidly reject allografts. While genetic disparities between donor and recipient are major determinants of the kinetics of transplant rejection, little is known about the contribution of environmental factors. Because colonized organs have worse transplant outcome than sterile organs, we tested the influence of host and donor microbiota on skin transplant rejection. Compared with untreated conventional mice, pretreatment of donors and recipients with broad-spectrum antibiotics (Abx) or use of germ-free (GF) donors and recipients resulted in prolonged survival of minor antigen-mismatched skin grafts. Increased graft survival correlated with reduced type I IFN signaling in antigen-presenting cells (APCs) and decreased priming of alloreactive T cells. Colonization of GF mice with fecal material from untreated conventional mice, but not from Abx-pretreated mice, enhanced the ability of APCs to prime alloreactive T cells and accelerated graft rejection, suggesting that alloimmunity is modulated by the composition of microbiota rather than the quantity of bacteria. Abx pretreatment of conventional mice also delayed rejection of major antigen-mismatched skin and MHC class II-mismatched cardiac allografts. This study demonstrates that Abx pretreatment prolongs graft survival, suggesting that targeting microbial constituents is a potential therapeutic strategy for enhancing graft acceptance.

Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants.

Dietary intervention with extensively hydrolyzed casein formula supplemented with Lactobacillus rhamnosus GG (EHCF+LGG) accelerates tolerance acquisition in infants with cow's milk allergy (CMA). We examined whether this effect is attributable, at least in part, to an influence on the gut microbiota. Fecal samples from healthy controls (n=20) and from CMA infants (n=19) before and after treatment with EHCF with (n=12) and without (n=7) supplementation with LGG were compared by 16S rRNA-based operational taxonomic unit clustering and oligotyping. Differential feature selection and generalized linear model fitting revealed that the CMA infants have a diverse gut microbial community structure dominated by Lachnospiraceae (20.5±9.7%) and Ruminococcaceae (16.2±9.1%). Blautia, Roseburia and Coprococcus were significantly enriched following treatment with EHCF and LGG, but only one genus, Oscillospira, was significantly different between infants that became tolerant and those that remained allergic. However, most tolerant infants showed a significant increase in fecal butyrate levels, and those taxa that were significantly enriched in these samples, Blautia and Roseburia, exhibited specific strain-level demarcations between tolerant and allergic infants. Our data suggest that EHCF+LGG promotes tolerance in infants with CMA, in part, by influencing the strain-level bacterial community structure of the infant gut.

Commensal bacteria protect against food allergen sensitization.

Environmentally induced alterations in the commensal microbiota have been implicated in the increasing prevalence of food allergy. We show here that sensitization to a food allergen is increased in mice that have been treated with antibiotics or are devoid of a commensal microbiota. By selectively colonizing gnotobiotic mice, we demonstrate that the allergy-protective capacity is conferred by a Clostridia-containing microbiota. Microarray analysis of intestinal epithelial cells from gnotobiotic mice revealed a previously unidentified mechanism by which Clostridia regulate innate lymphoid cell function and intestinal epithelial permeability to protect against allergen sensitization. Our findings will inform the development of novel approaches to prevent or treat food allergy based on modulating the composition of the intestinal microbiota.

TLR4 regulates IFN-γ and IL-17 production by both thymic and induced Foxp3+ Tregs during intestinal inflammation.

Tregs play a crucial role in the maintenance of intestinal immune homeostasis. However, significant numbers of Foxp3(+) Tregs accumulate in the inflamed lesions in experimental colitis and in IBD patients. Treg production of the proinflammatory cytokines IFN-γ and/or IL-17 may arguably explain their ineffectiveness in suppressing intestinal inflammation. However, it remains unknown whether iTreg and tTreg produce proinflammatory cytokines and how TLR signaling regulates this process. Here, we found that Foxp3(+)Tregs were increased in the intestines of B6.TLR4(-/-) and B6.IL-10(-/-) mice when compared with WT B6 mice. TLR4(-/-) and IL-10(-/-) resulted in more Tregs within inflamed intestines. The majority of Foxp3(+) Tregs in the spleen was Helios(+)Nrp1(+), whereas most Foxp3(+) Tregs in the intestinal LP were Helios(-)Nrp1(-). More Helios(+)Nrp1(+) Tregs expressed IFN-γ and/or IL-17 than did Helios(-)Nrp1(-) Tregs in the spleen and intestine, which was increased with TLR4(-/-). TLR4 signaling in T cells and APCs inhibited Foxp3(+) induction via MyD88-dependent, TRIF-independent pathways, which was negatively regulated by SOCS3. Collectively, these data demonstrate Helios(+)Nrp1(+) tTregs and Helios(-)Nrp1(-) iTregs produce proinflammatory cytokines in the intestines during inflammation, which was regulated by TLR4 signaling.

Microbial regulation of allergic responses to food.

The incidence of food allergy in developed countries is rising at a rate that cannot be attributed to genetic variation alone. In this review, we discuss the environmental factors that may contribute to the increasing prevalence of potentially fatal anaphylactic responses to food. Decreased exposure to enteric infections due to advances in vaccination and sanitation, along with the adoption of high-fat (Western) diets, antibiotic use, Cesarean birth, and formula feeding of infants, have all been implicated in altering the enteric microbiome away from its ancestral state. This collection of resident commensal microbes performs many important physiological functions and plays a central role in the development of the immune system. We hypothesize that alterations in the microbiome interfere with immune system maturation, resulting in impairment of IgA production, reduced abundance of regulatory T cells, and Th2-skewing of baseline immune responses which drive aberrant responses to innocuous (food) antigens.

Regulation of the polymeric immunoglobulin receptor in intestinal epithelial cells by Enterobacteriaceae: implications for mucosal homeostasis.

The commensal microbiota of the human colon profoundly impacts host gene expression and mucosal homeostasis. Secretory IgA antibodies, which influence the composition of the intestinal microbiota and provide immunity against pathogens, are transported across intestinal epithelial cells (IEC) by the polymeric immunoglobulin receptor (pIgR). To compare the effects of different colonic bacteria on pIgR expression, the human IEC line HT-29 was stimulated with various species representing the 4 major phyla of colonic bacteria. Only bacteria from the family Enterobacteriaceae (phylum Proteobacteria) induced expression of pIgR and other target genes of bacterial pattern recognition receptors. HT-29 cells responded to purified ligands for Toll-like receptor (TLR)4 but not TLR2. Expression of pIgR and transport of IgA were significantly reduced in colons of mice deficient in the TLR adaptor MyD88, consistent with a role for TLR signaling in the regulation of pIgR by colonic bacteria. Induction of pIgR expression in HT-29 cells required NF-kappaB signaling but not MAPK signaling, in contrast to the requirement for both NF-kappaB and MAPK signaling for induction of pro-inflammatory genes. These results suggest that commensal Enterobacteriaceae may promote intestinal homeostasis by enhancing pIgR expression in IEC.

Toll-like receptor 4-mediated regulation of spontaneous Helicobacter-dependent colitis in IL-10-deficient mice.

BACKGROUND & AIMS: The commensal microbiota is believed to have an important role in regulating immune responsiveness and preventing intestinal inflammation. Intestinal microbes produce signals that regulate inflammation via Toll-like receptor (TLR) signaling, but the mechanisms of this process are poorly understood. We investigated the role of the anti-inflammatory cytokine interleukin (IL)-10 in this signaling pathway using a mouse model of colitis. METHODS: Clinical, histopathologic, and functional parameters of intestinal inflammation were evaluated in TLR4(-/-), IL-10(-/-), and TLR4(-/-) x IL-10(-/-) mice that were free of specific pathogens and in TLR4(-/-) x IL-10(-/-) mice following eradication and reintroduction of Helicobacter hepaticus. Regulatory T-cell (Treg) function was evaluated by crossing each of the lines with transgenic mice that express green fluorescent protein under control of the endogenous regulatory elements of Foxp3. Apoptotic cells in the colonic lamina propria were detected by a TUNEL assay. RESULTS: TLR4-mediated signals have 2 interrelated roles in promoting inflammation in TLR4(-/-) x IL-10(-/-) mice. In the absence of TLR4-mediated signals, secretion of proinflammatory and immunoregulatory cytokines is dysregulated. Tregs (Foxp3(+)) that secrete interferon-gamma and IL-17 accumulate in the colonic lamina propria of TLR4(-/-) x IL-10(-/-) mice and do not prevent inflammation. Aberrant control of epithelial cell turnover results in the persistence of antigen-presenting cells that contain apoptotic epithelial fragments in the colonic lamina propria of Helicobacter-infected TLR4(-/-) mice. CONCLUSIONS: In mice that lack both IL-10- and TLR4-mediated signals, aberrant regulatory T-cell function and dysregulated control of epithelial homeostasis combine to exacerbate intestinal inflammation.