ALBERT

Description: Bortezomib, a therapeutic agent for multiple myeloma (MM) and mantle cell lymphoma, suppresses proteosomal degradation leading to substantial changes in cellular transcriptional programs and ultimately resulting in apoptosis. Transcriptional regulators required for bortezomib-induced apoptosis in MM cells are largely unknown. Using gene expression profiling, we identified 36 transcription factors that displayed altered expression in MM cells treated with bortezomib. Analysis of a publically available database identified Kruppel-like family factor 9 (KLF9) as the only transcription factor with significantly higher basal expression in MM cells from patients who responded to bortezomib compared with nonresponders. We demonstrated that KLF9 in cultured MM cells was up-regulated by bortezomib; however, it was not through the induction of endoplasmic reticulum stress. Instead, KLF9 levels correlated with bortezomib-dependent inhibition of histone deacetylases (HDAC) and were increased by the HDAC inhibitor LBH589 (panobinostat). Furthermore, bortezomib induced binding of endogenous KLF9 to the promoter of the proapoptotic gene NOXA. Importantly, KLF9 knockdown impaired NOXA up-regulation and apoptosis caused by bortezomib, LBH589, or a combination of theses drugs, whereas KLF9 overexpression induced apoptosis that was partially NOXA-dependent. Our data identify KLF9 as a novel and potentially clinically relevant transcriptional regulator of drug-induced apoptosis in MM cells.

Description: Introduction: Hematopoietic stem cell transplantation (HSCT) is associated with severe intestinal microbiota injury as a consequence of broad spectrum antibiotics, mucosal damage from conditioning, and dietary changes. Intestinal dysbiosis has been correlated with adverse outcomes post-transplant. We aimed to characterize and compare the patterns of microbiota injury that occur post autologous and allogeneic transplant. We characterized the specific risk factors associated with decreased microbial diversity in HSCT, and defined specific bacterial taxa that are enriched or depleted in patients based on the type of transplant and various other risk factors Methods: Stool samples from 35 patients (pts) undergoing HSCT were collected immediately pre-conditioning, at day 14, 28, and 100 post-transplant. 17 pts underwent an autologous (auto-) and 18 pts underwent an allogeneic transplant (allo-HSCT). The stool microbiome was characterized by sequencing of the V3V4 16s rRNA region; a- and b-diversity were determined and differential abundance analysis of OTUs was performed using DESeq2. Results: In the auto-HSCT cohort 70% of pts had plasma cell dyscrasia, and 24% had non-Hodgkin lymphoma. 83% of pts in the allo-HSCT cohort had a myeloid neoplasm, and 17% had a T cell neoplasm. 33% of pts undergoing allo-HSCT developed grade 2-4 GVHD within 100 days and 56% developed GVHD within 6 months. The pre-transplant intestinal microbiome α-diversity was significantly higher in pts undergoing auto-HSCT compared to allo-HSCT (Fig 1). The etiology for this was likely multifactorial. More pts in the allo-HSCT cohort had received antibiotics within 30 days prior to transplant, compared to the auto-HSCT cohort (56% vs 18%). Pts in the allo-HSCT cohort had a higher proportion of VRE or MDR Gram-negative bacteria on screening cultures (44% vs 29%), which may be associated with domination of certain species. In both the allo- and auto-HSCT cohorts, the α-diversity significantly decreased at days 14 and 28, but recovered to pre-transplant levels by day 100. Pts in the auto-HSCT cohort displayed faster recovery kinetics, and had a significantly higher α-diversity at day 28 compared to the allo-HSCT cohort (Fig 1). The microbiome recovery in the auto-HSCT cohort was consistent across the whole group and was characterized by a much smaller variance compared to the allo-HSCT cohort. This may reflect lower total antibiotic exposure in the auto-HSCT cohort (Fig 2). Other factors specific to the allo-HSCT cohort were the occurrence of GVHD and the use of TBI, both of which were associated with decrease in α-diversity in our population (Fig 3). Of note, Faecalibacterium prausnitzii was found to have a 〉20 log reduction in both the auto and allo-HSCT cohorts. It is a commensal gut bacterium, and its depletion has previously been reported in inflammatory bowel disease. There is very limited data on this bacterium in the peri-transplant setting. It has an anti-inflammatory effect on the intestinal microenvironment through production of butyrate, inhibition of NF-kB, upregulation of regulatory T cell production, and release of metabolites that enhance intestinal barrier function (Lopez-Siles et al, ISME 2017). Transplantation of F. prausnitzii has been shown to decrease inflammation in mice models of colitis (Sokol 2009). We then evaluated the association between microbiota injury and GVHD. α-diversity was significantly decreased at day 14 and day 28 in pts who developed GVHD within 100 days. In differential abundance analysis of samples from pts who developed acute GVHD within 6 months, there was a 〉20 log reduction in the Bifidobacterium, Blautia, and Eubacterium Dolichum. Decrease in Blautia has previously been associated with increased GVHD related mortality. Bifidobacterium and Eubacterium Dolichum have not been reported in association with GVHD in humans before. All three bacterial taxa reported here are involved in production of short chain fatty acids, which may provide a common protective mechanism. In summary, this study showed that pts undergoing autologous transplant have a healthier pre-transplant microbial diversity, and that microbiota injury heals quicker after transplant. We have identified TBI as a risk factor for microbiome injury, which has not been previously studied in the context of HSCT. We also identify previously unreported bacterial taxa whose depletion is strongly associated with GVHD. Disclosures Assal: Incyte corporation: Consultancy, Research Funding; boston biomedical: Consultancy. Uhlemann:Merk: Research Funding; GSK: Research Funding; Allergan: Research Funding. Reshef:Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Atara: Consultancy, Research Funding; Magenta: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Incyte: Consultancy, Research Funding; Shire: Research Funding; BMS: Consultancy.

Description: The gastrointestinal (GI) tract is a major target in GVHD. Conditioning-induced damage and mucosal barrier disruption are important factors in GVHD, however therapies targeting these processes have not been identified. Glucagon-like-peptide 2 (GLP-2) is an enterocyte-specific growth factor produced by L cells that has regenerative potential in models of GI damage. Its impact on the mucosal immune system has not been elucidated. We sought to examine the therapeutic and immunologic effect of GLP-2 in murine GVHD. We employed a major MHC-mismatched GVHD model (C57BL/6J → BALB/cJ). Mice were treated with 800nmol/kg/day of Elsiglutide (a GLP-2 analogue, provided by Helsinn) or vehicle beginning on D+1 for 30 days. Treatment with GLP-2 significantly improved survival and GVHD scores (Fig. 1A), while increasing small intestine mass and villi length (Fig 1B). GLP-2 also reduced T-cell infiltration into the jejunum (Fig. 1C). Analysis of intestinal immune cells by 28-color flow cytometry revealed dramatic differences between treatment groups in both myeloid- and T-cells. On D+14, GLP-2 led to an increased proportion of donor CSF-1R+ macrophages in the lamina propria (LP) (Fig. 2A) - cells that support the maintenance of the intestinal stem cell niche (Sehgal, Nat Commun, 2018). On D+21 the LP donor myeloid compartment was further altered, especially in MHC IIlow F4/80+ CD64+ macrophages (Fig. 2B, C). Here GLP-2 treatment expanded macrophages with lower expression of the co-stimulatory molecules CD80 and CD86 as well as the phagocytic marker CD206, whilst increasing the inhibitory molecule SIRPα, consistent with a tolerogenic phenotype. GLP-2 treatment also increased CX3CR1 expression on MHC IIlow macrophages with reduced Ly6C - a phenotype associated with physiologic macrophage maturation and linked to the resolution of colitis (Zigmond, Immunity, 2012). Vehicle-treated mice, conversely, had predominance of Ly6Chigh MHC IIlow LP macrophages reminiscent of an early infiltrating phenotype and near absence of mature macrophages, suggesting an impaired monocyte-macrophage transition that was restored by GLP-2. In addition, GLP-2 treatment led to significant changes in donor intraepithelial lymphocytes on D+21 (Fig. 2D), where CD8 T cells exhibited decreased CD27, CD103 and CXCR3 expression but higher PD-1, suggesting less activation. To assess potential mechanisms for the differences in macrophage and T-cell phenotype, we examined the impact of GLP-2 on the intestinal microbiota. A syngeneic BALB/cJ model was used to explore the effects of GLP-2 independent of GVHD. Stool samples from D+0, D+14, and D+28 were subjected to 16S rRNA sequencing. Vehicle-treated mice had distinct β-diversity clusters at all time-points, showing a transplant effect on the microbiota (Fig. 3A). GLP-2-treated mice had near-complete cluster overlap between D+0 and D+14, suggesting attenuation of the impact of conditioning. GLP-2 treated mice were significantly enriched for Akkermansia muciniphila and Bacteroidales S24-7 family at D+14 and D+28 (Fig. 3B). These taxa have been associated with anti-inflammatory properties and A. muciniphila abundance is linked to epithelial mucin production, which is increased by GLP-2. We then assessed the role of microbial communities in the protective effect of GLP-2 by conducting an allogeneic transplant with 3 caging conditions; 1) vehicle and GLP-2 treated mice caged together, 2) caged separately, or 3) caged separately plus oral antibiotics. We observed a clear cage effect where co-housing the treatment groups improved the survival of vehicle treated mice (Fig. 3C), suggesting transferal of the therapeutic effect via the microbiome. Antibiotic administration also dampened the beneficial effect of GLP-2. Finally, we conducted a GvL experiment by co-transplanting Luc-A20 and monitoring tumor progression via bioluminescence imaging. Both GLP-2 and vehicle-treated mice eliminated the tumor, whereas mice receiving T-cell depleted bone marrow showed tumor progression (Fig. 3D). In summary, our results demonstrate high therapeutic potential of GLP-2 in GVHD. GLP-2 administration led to reduced mortality, modified the microbiome and altered the intestinal immune response to a more tolerogenic state. This novel mechanism sheds light on the role of the enteroendocrine system in maintaining gut homeostasis and sets the stage for therapeutic clinical trials. Figure 1 Disclosures Uhlemann: Allergan: Research Funding; GSK: Research Funding; Merck: Research Funding. Reshef:Gilead: Consultancy; Magenta: Consultancy; Novartis: Consultancy; Monsato: Consultancy; Atara: Consultancy; BMS: Consultancy.