Role of integrins in the metastatic spread of high-grade serous ovarian cancer

Purpose Integrins may be involved in the metastatic spread of high-grade serous ovarian cancer (HGSOC) which determines the therapeutical approach and prognosis. We investigated the integrin expression in primary tumor and metastases of advanced HGSOC. Methods The expression of integrin α2, α4, α5, α6, and β1 was assessed by immunostaining in tumor samples of the ovary, omentum, and peritoneum of each patient. Differences in integrin expression among tumor localizations and their association with clinicopathological parameters were examined by Fisher’s exact test. The impact of integrin expression on progression-free survival (PFS) and overall survival (OS) was examined by Cox regression and Kaplan–Meier analyses. Results Hundred and thirteen tumor samples of 40 HGSOC patients were examined. The expression of the integrins did not differ between the three tumor localizations (all p values > 0.05) with the exception of high expression of integrin α4 in primary tumor and omentum (52.5% versus 47.5%, p = 0.008) and primary tumor and peritoneum (52.5% versus 47.5%, p = 0.050). High expression of integrin α4 in peritoneum was associated with poorer PFS (HR 2.02 95% CI 1.01–4.05, p = 0.047), younger age (p = 0.047), and death (p = 0.046). Median PFS in patients with high expression of integrin α4 was 13.00 months, whereas median PFS in patients without high expression of integrin α4 was 21.00 months (p = 0.040). Expression of other integrins did not correlate with PFS or OS. Conclusion Expression of integrin α4 may be altered during the metastatic spread of HGSOC and affect prognosis, whereas expression of integrin α2, α5, α6, and β1 did not reveal any prognostic value.

In vivo self-assembled siRNA: a new modality for combination therapy of ulcerative colitis

Given the complex nature of ulcerative colitis (UC), combination therapy targeting multiple pathogenic genes and pathways of UC may be required. Unfortunately, current therapeutic strategies based on independent chemical compounds or monoclonal antibodies are not applicable for combination therapy of UC. Here, we developed a synthetic biology strategy that integrates the naturally existing exosome-circulating system with artificial genetic circuits for self-assembly and delivery of multiple siRNAs for the combination therapy of UC. Intravenous injection of genetic circuit (in the form of DNA plasmid) designed for inhibition of TNF-α, B7-1 and integrin α4 successfully reprogrammed the host liver to direct the self-assembly of TNF-α, B7-1 and integrin α4 siRNA into secretory exosomes. The multitargeted genetic circuit could rapidly relieved intestinal inflammation and exert a synergistic therapeutic effect against UC through suppressing the proinflammatory cascade in colonic macrophages, inhibiting the costimulatory signal to T cells and blocking T cell homing to sites of inflammation. More importantly, we designed an AAV-driven genetic circuit to induce substantial and lasting inhibition of TNF-α, B7-1 and integrin α4. Overall, this study established a feasible combination therapeutic strategy for UC, which is superior to the conventional biological therapies requiring.

O-GlcNAcylation homeostasis controlled by calcium influx channels regulates multiple myeloma dissemination

Background Multiple myeloma (MM) cell motility is a critical step during MM dissemination throughout the body, but how it is regulated remains largely unknown. As hypercalcemia is an important clinical feature of MM, high calcium (Ca2+) and altered Ca2+ signaling could be a key contributing factor to the pathological process. Methods Bioinformatics analyses were employed to assess the clinical significance of Ca2+ influx channels in clinical specimens of smoldering and symptomatic MM. Functional and regulatory roles of influx channels and downstream signaling in MM cell migration and invasion were conducted and experimental MM dissemination was examined in a xenograft mouse model using in vivo live imaging and engraftment analysis. Results Inhibition of TRPM7, ORAI1, and STIM1 influx channels, which are highly expressed in MM patients, and subsequent blockage of Ca2+ influx by CRISPR/Cas9 and small molecule inhibitors, effectively inhibit MM cell migration and invasion, and attenuate the experimental MM dissemination. Mechanistic studies reveal a nutrient sensor O-GlcNAcylation as a downstream regulator of Ca2+ influx that specifically targets cell adhesion molecules. Hyper-O-GlcNAcylation following the inhibition of Ca2+ influx channels induces integrin α4 and integrin β7 downregulation via ubiquitin-proteasomal degradation and represses the aggressive MM phenotype. Conclusions Our findings unveil a novel regulatory mechanism of MM cell motility via Ca2+ influx/O-GlcNAcylation axis that directly targets integrin α4 and integrin β7, providing mechanistic insights into the pathogenesis and progression of MM and demonstrating potential predictive biomarkers and therapeutic targets for advanced MM.

Abnormal promoter DNA hypermethylation of the integrin, nidogen, and dystroglycan genes in breast cancer

Cell transmembrane receptors and extracellular matrix components play a pivotal role in regulating cell activity and providing for the concerted integration of cells in the tissue structures. We have assessed DNA methylation in the promoter regions of eight integrin genes, two nidogen genes, and the dystroglycan gene in normal breast tissues and breast carcinomas (BC). The protein products of these genes interact with the basement membrane proteins LAMA1, LAMA2, and LAMB1; abnormal hypermethylation of the LAMA1, LAMA2, and LAMB1 promoters in BC has been described in our previous publications. In the present study, the frequencies of abnormal promoter hypermethylation in BC were 13% for ITGA1, 31% for ITGA4, 4% for ITGA7, 39% for ITGA9, 38% for NID1, and 41% for NID2. ITGA2, ITGA3, ITGA6, ITGB1, and DAG1 promoters were nonmethylated in normal and BC samples. ITGA4, ITGA9, and NID1 promoter hypermethylation was associated with the HER2 positive tumors, and promoter hypermethylation of ITGA1, ITGA9, NID1 and NID2 was associated with a genome-wide CpG island hypermethylated BC subtype. Given that ITGA4 is not expressed in normal breast, one might suggest that its abnormal promoter hypermethylation in cancer is non-functional and is thus merely a passenger epimutation. Yet, this assumption is not supported by our finding that it is not associated with a hypermethylated BC subtype. ITGA4 acquires expression in a subset of breast carcinomas, and methylation of its promoter may be preventive against expression in some tumors. Strong association of abnormal ITGA4 hypermethylation with the HER2 positive tumors (p = 0.0025) suggests that simultaneous presence of both HER2 and integrin α4 receptors is not beneficial for tumor cells. This may imply HER2 and integrin α4 signaling pathways interactions that are yet to be discovered.

Characterizing the Motility of Chemotherapeutics-Treated Acute Lymphoblastic Leukemia Cells by Time-Lapse Imaging

Drug resistance is an obstacle in the therapy of acute lymphoblastic leukemia (ALL). Whether the physical properties such as the motility of the cells contribute to the survival of ALL cells after drug treatment has recently been of increasing interest, as they could potentially allow the metastasis of solid tumor cells and the migration of leukemia cells. We hypothesized that chemotherapeutic treatment may alter these physical cellular properties. To investigate the motility of chemotherapeutics-treated B-cell ALL (B-ALL) cells, patient-derived B-ALL cells were treated with chemotherapy for 7 days and left for 12 h without chemotherapeutic treatment. Two parameters of motility were studied, velocity and migration distance, using a time-lapse imaging system. The study revealed that compared to non-chemotherapeutically treated B-ALL cells, B-ALL cells that survived chemotherapy treatment after 7 days showed reduced motility. We had previously shown that Tysabri and P5G10, antibodies against the adhesion molecules integrins α4 and α6, respectively, may overcome drug resistance mediated through leukemia cell adhesion to bone marrow stromal cells. Therefore, we tested the effect of integrin α4 or α6 blockade on the motility of chemotherapeutics-treated ALL cells. Only integrin α4 blockade decreased the motility and velocity of two chemotherapeutics-treated ALL cell lines. Interestingly, integrin α6 blockade did not affect the velocity of chemoresistant ALL cells. This study explores the physical properties of the movements of chemoresistant B-ALL cells and highlights a potential link to integrins. Further studies to investigate the underlying mechanism are warranted.

Cell Adhesion of ALL to Stromal Cells May Mediate CAR T-Cell Resistance: A Novel Escape Mechanism for Immunotherapy

Backgroud: Despite advances in therapy and improved survival, relapsed and refractory B-cell precursor acute lymphoblastic leukemia (r/r BCP-ALL) in pediatric and adult patients still remains a problem. Chimeric antigen receptor T cells against CD19 (CD19 CAR T) show promising results in patients with r/r BCP-ALL. However, relapse of the disease still occurs with appreciable frequency even with this novel therapy. As a significant number of relapses post-CAR T lack surface CD19 expression, further CD19-directed therapy is not an option for these cases. Hypothesis: Sometimes despite CAR T engraftment and establishment of B-cell aplasia, relapse still occurs. We hypothesized that, similarly to cell adhesion mediated chemotherapeutic drug resistance (CAM-DR), cell adhesion mediated CAR T-cell resistance (CAM-CART-R) can contribute to relapse of ALL. Results: To test our hypothesis, primary ALL cells were treated with CD19 CAR T cells either with murine calvaria-derived bone marrow stromal cells, OP9, or cultured only with media in short term cultures. We observed B-ALL cells treated with CD19 CAR T on OP9 has 10-20% higher viability compared to B-ALL and CD19 CAR T co-culture in medium alone, supporting the notion of CAM-CART-R. We also determined that soluble factors in OP9 primed medium may contribute to CAM-CART-R. However, the direct stromal contact mediated significant protection again CAR T induced apoptosis of B-ALL cells. To determine the molecular mechanisms underlying the survival promoting effects of stromal cells on CD19-, these cells were starved in serum-free media for 4hours and then treated with PI3Kδ inhibitor CAL-101 or DMSO and co-cultured with OP9 cells for 1 hour. We found that p-Akt is upregulated by stromal contact in CD19-negative B-ALL cells post-CAR T therapy and that PI3Kδ inhibition using can downregulate p-Akt in CD19-negative B-ALL patients. Critically, we investigated whether CD19 CAR T cells were functional under these conditions. For this purpose, we determined if stromal contact of ALL cells or stromal contact of CAR T cells changes the intracellular cytokine milieu of CD19 CAR T cells and found that intracellular IL-6, TNF- α and IFN-γ were reduced upon stromal contact supporting our hypothesis of a role of stromal cells in CAM-CART-R. We also determined that immune checkpoints molecules on T cells are unaffected by OP9 cells. Despite the reduction of cytokine level in T cells upon co-culture with B-ALL cells on OP9, PD-1, TIM-3 and LAG3 expression on CD19 CAR T cells after 2 days of co-culture was not altered as determined by flow cytometry. Resistance of ALL cells to CD19 CART cells was not mediated through checkpoint inhibition, since the PD-1/PD-L1 inhibitor Nivolumab failed to enhance ALL killing. Phenotypic profiling of thirteen cases of primary ALL relapse post-CD19 CAR T cell therapy showed high expression of adhesion molecules including integrin α4. Phenotypic analysis also revealed high expression of integrins is retained in primary ALL cells after CD19 knockout in one case. To explore possible solutions to overcome CAM-CART-R, we examined a strategy of blocking specifically integrin α4. We have previously shown that blocking integrin α4 can de-adhere CD19-negative B-ALL relapse post-CAR T cell therapy from their respective counter-ligands in vitro and that these cells can benefit from integrin blocking therapy in vivo. We have now confirmed this in NSG mice injected with CD19-negative B-ALL cells from a patient with post-CAR T cell relapse. Mice were treated intraperitoneally (n=6/group) with total immunoglobulin (Ig) control or humanized anti-human integrin α4 antibody Natalizumab (NZM). As a result, Natalizumab monotherapy significantly prolonged survival of leukemic mice compared to control Ig group (66 days (Ig) vs 85 days (NZM) p<0.005). Further combination treatments with chemotherapy are in progress. Conclusion: In summary, our data indicate that similarly to CAM-DR, CAM-CART-R can occur resulting in relapse of ALL. Targeting adhesion molecules may be a new approach to treat or prevent relapse following CD19 CAR T cell therapy for . Disclosures Ahmed: CellMedica: Other: Royalties; Celgene: Other: Royalties; Adaptimmune: Membership on an entity's Board of Directors or advisory committees. Babak:Simurx. Inc: Other: Founder . Pulsipher:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Lecture; Bellicum: Consultancy; Miltenyi: Research Funding; Medac: Honoraria. Wayne:AbbVie: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Servier: Consultancy; Spectrum Pharmaceuticals: Consultancy, Research Funding. Abdel-Azim:Adaptive: Research Funding.