Purpose Patients with advanced stages of MCL have a poor prognosis after standard therapies. MCL cells in those patients often spread into tissues other than lymph nodes, such as the bone marrow. Apart from directed migration and homing, there is little understanding of the function of the CXCR4/SDF1 signaling axis in MCL. In this report, we aim to understand mechanisms of MCL cell survival in the bone marrow. Experimental Design For comprehensive analyses of MCL interactions with bone marrow stromal cells we have generated gene knock out cells using CRISPR-CAS9 system and gene knock down cells to reveal novel roles of the CXCR4/SDF-1 signaling. Results CXCR4 silencing in MCL cells led to a significant reduction in proliferation, cell adhesion to bone marrow stromal cells and colony formation in PHA-LCM methylcellulose medium, which were reversed upon the addition of SDF-1 neutralizing antibodies. In addition, tracking MCL cell engraftment in vivo revealed that quiescent MCL cells are significantly reduced in the bone marrow upon CXCR4 silencing, indicating that CXCR4/SDF-1 signaling is required for the survival and maintenance of the quiescent MCL cells. Further analysis revealed novel mechanisms of ROS induced CXCR4/SDF-1 signaling that stimulate autophagy formation in MCL cells for their survival. Conclusions Our data, for the first time, revealed new roles of the CXCR/SDF-1 signaling axis on autophagy formation in MCL, which further promoted their survival within the bone marrow microenvironment. Targeting the CXCR4/SDF-1/autophagy signaling axis may contribute to an enhanced efficacy of current therapies.

Reduced PAX5 levels play important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5− MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to bone marrow stromal cells, and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb and to PI3 kinase/mTOR and pSTAT3 pathway hyperactivation. Upon PAX5 silencing, the MCL cells displayed upregulated IL-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5−MCL cells are highly drug-resistant compared to PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B cell cancer and encourage future efforts to design effective therapies against MCL.

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many types of cancer cells. TRAIL is considered a therapeutic target, therefore, it was of interest to examine molecular mechanisms that may modulate sensitivity to TRAIL signaling in prostate cancer cells. LNCaP cells were found to be relatively resistant to TRAIL induced cell death while PC3 cells were sensitive. PI3-kinase (PI3 K) inhibitors were able to render LNCaP cells sensitive to TRAIL but conferred resistance to PC3 cells. PI3 K inhibitors were associated with an increase in p21(waf1, cip1) expression in PC3 cells where as p21 decreases in LNCaP cells suggesting that p21 may impart TRAIL resistance. Since androgen receptor (AR) signaling can be modulated by AKT, and p21 is an AR responsive gene, the impact of PI3 K inhibition on TRAIL sensitivity was evaluated in AR transfected PC3 cells (PC3AR). The expression of AR was significantly downregulated by PI3 K inhibition in LNCaP cells, which have an intact AR signaling axis. PC3AR cells expressed higher levels of p21 protein and were relatively resistant to TRAIL compared to control cells. Finally, using adenoviral p21 gene transfer we directly demonstrated that p21 can confer resistance to TRAIL-induced cell death. These results suggest that TRAIL resistance is not regulated simply by a PI3 K/AKT survival pathway associated with inactivating PTEN mutations but may also be modulated by downstream AR responsive targets such as p21. These findings may have significant clinical implications for the utility of TRAIL in the management of prostate cancer.

<div>Abstract<p><b>Purpose:</b> Patients with advanced stages of mantle cell lymphoma (MCL) have a poor prognosis after standard therapies. MCL cells in those patients often spread into tissues other than lymph nodes, such as the bone marrow. Apart from directed migration and homing, there is little understanding of the function of the CXCR4/SDF-1 signaling axis in MCL. In this report, we aim to understand mechanisms of MCL cell survival in the bone marrow.</p><p><b>Experimental Design:</b> For comprehensive analyses of MCL interactions with bone marrow stromal cells, we have generated gene knockout cells using CRISPR-CAS9 system and gene knockdown cells to reveal novel roles of the CXCR4/SDF-1 signaling.</p><p><b>Results:</b> CXCR4 silencing in MCL cells led to a significant reduction in proliferation, cell adhesion to bone marrow stromal cells, and colony formation in PHA-LCM methylcellulose medium, which were reversed upon the addition of SDF-1–neutralizing antibodies. In addition, tracking MCL cell engraftment <i>in vivo</i> revealed that quiescent MCL cells are significantly reduced in the bone marrow upon <i>CXCR4</i> silencing, indicating that CXCR4/SDF-1 signaling is required for the survival and maintenance of the quiescent MCL cells. Further analysis revealed novel mechanisms of ROS-induced CXCR4/SDF-1 signaling that stimulate autophagy formation in MCL cells for their survival.</p><p><b>Conclusions:</b> Our data, for the first time, revealed new roles of the CXCR/SDF-1 signaling axis on autophagy formation in MCL, which further promoted their survival within the bone marrow microenvironment. Targeting the CXCR4/SDF-1/autophagy signaling axis may contribute to an enhanced efficacy of current therapies. <i>Clin Cancer Res; 22(1); 187–99. ©2015 AACR</i>.</p></div>

<p>Figure S1: FACS analyses of CXCR4 silenced in Jeko, SP53 and Z138 MCL cells. GFP-positive cells were sorted after infection and CXCR4 expression was evaluated using FACS analyses. 94-74% reduction was noted in all knock down cells. Figure S2: Migration of MCL cells to SDF-1 was effectively decreased after CXCR4 silencing. GFP-positive controls or CXCR4 silenced MCL cells (105) were placed in upper chambers with different concentrations of SDF-1 in lower chambers and the cells were counted in the lower chamber after 24 hours. Figure S3: CXCR4 silenced cells showed reduction of migration toward the medium harvested from HS27a culture. Medium from HS27a was harvested after 3 days and 600 mL was added to transwell. GFP-positive controls or CXCR4 silenced MCL cells (Jeko, SP53, Z138 and REC1, 105) were placed in upper chambers with HS27a medium in lower chambers and cells were counted in the lower chambers after 24 hours. Figure S4: SDF-1 protects the MCL cells from starvation induced cell death but neutralized antibodies were reversed the effect of protection. MCL cells (1x106) were cultured in low serum for three days and SDF-1 (10ng/ml) or neutralizing SDF-1 antibodies were added to the culture. Cell death were evaluated based on 7-AAD/Annexin V staining using FACS. Figure S5: Colonies from PHA-LCM medium was harvested and light chain restriction was analyzed using fix and perm cell permeabilization kit (Invitrogen). Colonies harvested from PHA-LCM medium were lambda restricted similar to the SP53 parent cells. Figure S6: CXCR4 silencing decreases the motility of MCL cells. We quantitatively measured the distances the CXCR4-silenced or control cells travelled over 2 days using a time-lapse microscopy. Distances (mm) traveled by CXCR4-silenced SP53 and REC1 cells, which were an average of results from 15 different wells, were significantly decreased compared to the control cells. Figure S7: Bortezomib IC50 was calculated using different MCL cells. IC50 value (the concentration of a drug that is required for 50% inhibition in vitro) was used to indicate the quantitative measure of the different cell killing effect of drugs. The Hill-Slope logistic model is used to calculate IC50 using MS Excel. Figure S8A&B: (A) HS27a stromal cells as well as HS27a media protect MCL cells from chemotherapeutic drug cytotoxicity. CD19+ cells were isolated from four different patient cells (105) and were cultured with HS27a stromal cells or 1 ml of 1:1 diluted HS27a medium. Cell viability was determined by the CellTiter-Blue® fluorometric assay (Promega) and was indicated as a ratio compared to cell viability without treatment. Bortezomib was serially diluted (0-40 nM) as indicated. The results represent the mean {plus minus} standard deviation of triplicates. *, P<0.05; **, P<0.005. P values were calculated using Student's t-test. (B) AMD3100 treatment abolishes the protective effects of the stromal cells against chemotherapeutic drugs. A CXCR4 antagonist, AMD3100 (40 mM), was incubated with MCL cells for 24 hours, and cell viability was determined by CellTiter-Blue. The results represent the mean {plus minus} standard deviation of triplicates. *, P<0.05, **, P<0.005; P values were calculated using Student's t-test. Figure S9: Bortezomib treatment induces CXCR4 expression in MCL cells. Bortezomib-resistant Mino and REC1 cells (106, 6-well plate) were treated with different doses of bortezomib (0-100 nM for 24 hours). CXCR4 expression was analyzed by FACS. Figure S10: ROS generated after bortezomib treatment induces CXCR4 upregulation in MCL cells. Bortezomib-resistant Mino and REC1 cells (106, 6-well plate) were treated with bortezomib (30 nM for 24 hours) with or without NAC (100 mM, 1 hour). CXCR4 expression was evaluated using FACS. Figure S11: Ibrutinib IC50 was calculated using different MCL cells. IC50 value (the concentration of a drug that is required for 50% inhibition in vitro) was used to indicate the quantitative measure of the different cell killing effect of drugs. The Hill-Slope logistic model is used to calculate IC50 using MS Excel. Figure S12: HS27a stromal cells as well as HS27a media protect MCL cells from Ibrutinib cytotoxicity. CD19+ cells were isolated from four different patient cells (105) and were cultured with HS27a stromal cells or 1 ml of 1:1 diluted HS27a medium. Cell viability was determined by the CellTiter-Blue® fluorometric assay (Promega) and was indicated as a ratio compared to cell viability without treatment. Ibrutinib was serially diluted (0-40 nM) as indicated. The results represent the mean {plus minus} standard deviation of triplicates. *, P<0.05; **, P<0.005. P values were calculated using Student's t-test. Figure S13: ROS weren't generated after Ibrutinib treatment. Jeko and SP53 cells (106, 6-well plate) were treated with Ibrutinib (0, 20, 40 mM for 24 hours) and ROS was measured using DCH-FDA (Sigma-Aldrich). ROS was measured at Ex/Em, 480/520 nm and then analyzed by flow cytometry. Figure S14: Ibrutinib treatment did not induce CXCR4 upregulation in MCL cells. Jeko and SP53 cells (106, 6-well plate) were treated with Ibrutinib (0, 20 and 40 mM) and CXCR4 expression was evaluated using FACS. Figure S15: A model diagram depicting observations made in the study. Ibrutinib treatment does not produce ROS as well as does not induce upregulation of CXCR4. Figure S16: Bortezomib (30 nM) was added to the MCL cells with or without lysosomal inhibitor chloroquine (50 mM), and autophagy induction was analyzed based on the LC3B-I to LC3B-II conversion as measured by immunoblots. Figure S17: Beclin1 was knocked down using lentivirus mediated shRNA in Jeko cells and Beclin1 levels were evaluated using immunoblots. GFP+ cells were selected followed by antibiotic selection to ensure knock down. Densitometer analysis showed a significant down-regualtion of beclin 1 in GFP+ Jeko cells compared to GFP+ scrambled shRNA infected Jeko cells. Figure S18: Jeko cells were cultured in low serum condition with AMD3100 to inhibit CXCR4 expression and autophagy expression was analyzed using immunoblots based on LC3-I to LC3-II conversion. Figure S19: Beclin 1 silencing does not change CXCR4 expression in MCL cells.</p>