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REPURPOSING PENFLURIDOL: AN OLD ANTIPSYCHOTIC DRUG THAT THERAPEUTICALLY TARGETS PROLACTIN RECEPTOR IN PANCREATIC CANCER Prasad Dandawate, Gaurav Fnu, Dharmalingam Subramaniam, Prabhu Ramamoorthy, Santanu Paul, Animesh Dhar, Sufi M. Thomas, Santimukul Santra, Ossama Tawfik, Scott J. Weir, Roy A. Jensen, Shrikant Anant Pancreatic cancer (PCa) is a major cause of cancer-related deaths in the United States with < 6% survival rate. PCa has poor prognosis, shows rapid progression and invasiveness, resistance to anticancer drug treatment and recurrence after surgery. Currently, Gemcitabine is the major drug for treatment of metastatic adenocarcinoma of the pancreas, but a tumor response rate is low. Moreover, combination treatments have only slightly improved survival rate. Hence, there is a need to find novel targets for therapeutic modulation in PCa. In this regard, we recently identified that the receptor for the pituitary hormone prolactin is overexpressed in pancreatic cancers, and in cell lines. When prolactin (PRL) binds its cognate receptor (PRLR), it induces various downstream events including the JAK-STAT and ERKMAPK pathways. In PCa cell lines, we observed that PRL treatment induces dose- and timedependent increased phosphorylation of JAK2, STAT3, and ERK1/2. While PRL did not affect cell proliferation, it induced pancosphere formation suggesting that PRL affects stem cells. Furthermore, there was an increase in the expression of cancer stem cell markers DCLK1 (doublecortin calmodulin-like kinase 1) and CD44. On the other hand, CRISPR/ Cas9-mediated knockout of PRLR resulted in cells resulted in lower spheroid formation capacity and decreased migration. There was also a reduction in phosphorylation of STAT3, AKT, and ERK. Based on these data, we conclude that PRL signaling is an excellent target for therapeutic manipulation. No small molecules exist for PRLR. Hence, we developed a homology model for the C-terminal intracellular region of the receptor and performed a virtual screening in silico with FDA approved drugs. One compound, a first generation antipsychotic drug penfluridol was found to interact with the region of the receptor that also binds site for JAK2. The compound has a long half-life and is used in the treatment of chronic schizophrenia and similar psychotic disorders. We experimentally confirmed penfluridol binding to PRLR with the cellular thermal shift CETSA assay. In addition, PCa cells treated with Penfluridol demonstrated reduced PRL-induced phosphorylation of STAT3 and ERK, and spheroid formation. Moreover, it induces cells to undergo autophagy by activating LC3B, p62 and increasing expression of autophagy-related genes ATG-5, -7 and -12. Finally, we performed in vivo proof of principle experiments in an orthotopic PCa model with UNKC-6141 cells injected into the pancreas in C57BL/6 mice. Penfluridol treatment significantly reduced the tumor burden, and suppressed the JAK/STAT and ERK pathways. Taken together, these data suggest that PRL signaling through its cognate PRLR receptor is critical for aggressive pancreatic cancer behavior and therefore may be an effective therapeutic targeting strategy.
Figure 1. FMT treatment outcome is associated with the transfer of Caudovirales bacteriophages. (a) Presence-absence heatmap of Caudovirales contigs in pre-FMT and post-FMT stool samples for 9 FMT recipients. Class I subjects are individuals with donor Caudovirales richness> recipient Caudovirales richness and cured with FMT, Class II and III are subjects with donor Caudovirales richness < recipient Caudovirales richness but II were cured while III relapsed. Only contigs with rarefied RPKM > 3 were shown. Red lines indicate contigs were transferred from donor. (b) Present percentage of donor transferred Caudovirales contigs in FMT recipients at last follow up. The size of the circle indicates the count of Caudovirales contigs transferred from donor. The color of the circle indicates the richness type of the recipient.
639b INFECTION WITH THE FUNGUS CANDIDA TROPICALIS SIGNIFICANTLY INCREASES THE SEVERITY OF COLITIS IN C57BL/6 MICE Luca Di Martino, Kristine-Ann Buela, Christopher Hager, Mahmoud Ghannoum, Wei Xin, Theresa T. Pizarro, Fabio Cominelli
649 IMMUNE CHECK POINT INHIBITOR TREATMENT FOR YOUNG PATIENTS WITH HYPERMUTANT CANCERS Carol A. Durno, Melyssa Aronson, Melissa Edwards, Simon Ling, Valérie Larouche, Annika Bronsema, Michael Osborn, Duncan Stearns, Kristina Cole, Michal Oren, Enrico Opocher, Gary E. Mason, Gregory Thomas, Magnus Sabel, Ben George, David Ziegler, Scott Lindhorst, Vanan Magimairajan, Normand Laperriere, David Samuel, Eric Bouffet, Uri Tabori
Background: Fungal infections represent a serious health hazard, especially for immunocompromised patients. Infections (candidiasis) caused by Candida spp. including C. tropicalis, have dramatically increased worldwide, indicating that this organism is an emerging pathogenic yeast. Our recent studies demonstrate that the abundance of pathogenic C. tropicalis is significantly higher in Crohn's disease (CD) patients than in their non-disease first-degree relatives. However, the role of C. tropicalis in exacerbating intestinal inflammation in patients with CD has not been fully elucidated. The aim of the present study was to determine the effect(s) of C. tropicalis infection on intestinal inflammation following acute intestinal injury induced by dextran sodium sulfate (DSS) in mice. Methods: Colitis was studied in C57BL/ 6 mice by endoscopy, histology, qPCR and flow cytometry. Mice were infected with C. tropicalis by oral gavage (1x107 cells/100 µl PBS) once daily for three days. Colitis was induced by administration of 2.5% DSS in drinking water for 7 days. After 14 days of recovery with only drinking water, mice were sacrificed and colons and mesenteric lymph nodes (MLN) were collected. Results: C57BL/6 mice infected with C. tropicalis showed no differences in colonoscopy scores at day 7 compared to control, uninfected mice. However, increased severity of colitis vs. uninfected mice (endoscopy score: 21 days 7.033 ± 0.204 (infected) vs. 5.575 ± 0.528 (uninfected), P<0.05, n=4-6/group) was noted at day 21. IFNγ was significantly upregulated in the colons of infected compared to uninfected mice by 4.5-fold (qPCR analysis: P<0.05, n=4-6/group). No differences were observed in TNF mRNA expression in both groups. Flow cytometry analysis showed that in MLN of infected mice there was a higher percentage of group 2 innate lymphoid cells (ILC2s) compared to uninfected mice (% ILC2: 10.28 ± 2.283 (infected) vs. 2.075 ± 0.340 (uninfected), P<0.05, n=4-6/group). Histological analysis of colitis showed a trend of higher scores in infected mice compared to uninfected mice (6.250 ± 1.153 (infected) vs. 4.750 ± 1.785 (uninfected), P=0.4905, n=4-6/group). Conclusion: Our data demonstrate that C. tropicalis may play a pro-inflammatory role in intestinal injury by exacerbating gut inflammation during the recovery phase of DSS-induced colitis. We speculate that infection with the fungus C. tropicalis may play a role in triggering flares during CD and that anti-fungal therapy may be beneficial in CD patients.
Background: Hypermutant malignancies in adults have shown a positive response to immune checkpoint inhibitor therapy. Patients with biallelic mismatch repair deficiency syndrome (BMMRD) have a unique cancer phenotype with hypermutation in the tumors. We postulated that young patients with inoperable hypermutated cancers respond to immune checkpoint inhibition therapy. Methods: Patients referred to the International Biallelic Mismatch Repair Deficiency Consortium were confirmed to have BMMRD, Lynch syndrome, POLE or POLD1 germline mutations based on the identification of germline mutations and/or immunohistochemical confirmation. Exome or whole genome sequencing were performed on available tumors. Immune check point inhibitor therapy (ICI) were offered to patients with hypermutant inoperable malignancies. Results: Eighty-one patients were identified with 119 tumors. Malignancies include brain (48%), colorectal (28%), hematological (16%), genitourinary (2%) and other (n=6%). Oligo-adenomatous colorectal polyposis occurred in 79% of patients screened. Classification of patients include BMMRD (n=69), Lynch syndrome (n=7), POLE (n=3) and under evaluation (n=2). Among the 69 BMMRD patients, PMS2 mutations were most frequent (n=42), followed by MSH6 (n=16), MSH2 (n=5), MLH1 (n=4), and EPCAM (n=2). The Lynch syndrome patients had MSH2 (n=4), MLH1 (n=2), and unknown (n=1) mutations. BMMRD brain tumors had the highest mutation loads (mean 17 740+/-7703 mutations per exome) mostly due to secondary polymerase mutations. BMMRD colorectal tumors were hypermutant (mean 1589+/-1043 mutations per exome). Twenty patients were treated with ICI therapy: 16 BMMRD and 4 Lynch syndrome. Mean age at primary cancer diagnosis was 11.6 years (SD +/- 7, range 1.8 to 27.4 years). Three patients had double primary cancer in different organs, two of which had distant metastatic disease. All 4 colorectal, the one genitourinary and 4 of 9 brain cancers had measureable clinical and radiological response to therapy with an overall survival at one year of 61%. Conclusion: Cancers in patients with BMMRD, POLE mutations and some Lynch cancers have a hypermutant tumor phenotype. These data are promising, particularly the response of the colorectal and genitourinary tumors to therapy. A large international clinical trial for management of hypermutant cancers has recently commenced and will provide further validation for these novel observations.
S-141
AGA Abstracts
AGA Abstracts
648
Figure 1. FMT treatment outcome is associated with the transfer of Caudovirales bacteriophages. (a) Presence-absence heatmap of Caudovirales contigs in pre-FMT and post-FMT stool samples for 9 FMT recipients. Class I subjects are individuals with donor Caudovirales richness> recipient Caudovirales richness and cured with FMT, Class II and III are subjects with donor Caudovirales richness < recipient Caudovirales richness but II were cured while III relapsed. Only contigs with rarefied RPKM > 3 were shown. Red lines indicate contigs were transferred from donor. (b) Present percentage of donor transferred Caudovirales contigs in FMT recipients at last follow up. The size of the circle indicates the count of Caudovirales contigs transferred from donor. The color of the circle indicates the richness type of the recipient.
639b INFECTION WITH THE FUNGUS CANDIDA TROPICALIS SIGNIFICANTLY INCREASES THE SEVERITY OF COLITIS IN C57BL/6 MICE Luca Di Martino, Kristine-Ann Buela, Christopher Hager, Mahmoud Ghannoum, Wei Xin, Theresa T. Pizarro, Fabio Cominelli
649 IMMUNE CHECK POINT INHIBITOR TREATMENT FOR YOUNG PATIENTS WITH HYPERMUTANT CANCERS Carol A. Durno, Melyssa Aronson, Melissa Edwards, Simon Ling, Valérie Larouche, Annika Bronsema, Michael Osborn, Duncan Stearns, Kristina Cole, Michal Oren, Enrico Opocher, Gary E. Mason, Gregory Thomas, Magnus Sabel, Ben George, David Ziegler, Scott Lindhorst, Vanan Magimairajan, Normand Laperriere, David Samuel, Eric Bouffet, Uri Tabori
Background: Fungal infections represent a serious health hazard, especially for immunocompromised patients. Infections (candidiasis) caused by Candida spp. including C. tropicalis, have dramatically increased worldwide, indicating that this organism is an emerging pathogenic yeast. Our recent studies demonstrate that the abundance of pathogenic C. tropicalis is significantly higher in Crohn's disease (CD) patients than in their non-disease first-degree relatives. However, the role of C. tropicalis in exacerbating intestinal inflammation in patients with CD has not been fully elucidated. The aim of the present study was to determine the effect(s) of C. tropicalis infection on intestinal inflammation following acute intestinal injury induced by dextran sodium sulfate (DSS) in mice. Methods: Colitis was studied in C57BL/ 6 mice by endoscopy, histology, qPCR and flow cytometry. Mice were infected with C. tropicalis by oral gavage (1x107 cells/100 µl PBS) once daily for three days. Colitis was induced by administration of 2.5% DSS in drinking water for 7 days. After 14 days of recovery with only drinking water, mice were sacrificed and colons and mesenteric lymph nodes (MLN) were collected. Results: C57BL/6 mice infected with C. tropicalis showed no differences in colonoscopy scores at day 7 compared to control, uninfected mice. However, increased severity of colitis vs. uninfected mice (endoscopy score: 21 days 7.033 ± 0.204 (infected) vs. 5.575 ± 0.528 (uninfected), P<0.05, n=4-6/group) was noted at day 21. IFNγ was significantly upregulated in the colons of infected compared to uninfected mice by 4.5-fold (qPCR analysis: P<0.05, n=4-6/group). No differences were observed in TNF mRNA expression in both groups. Flow cytometry analysis showed that in MLN of infected mice there was a higher percentage of group 2 innate lymphoid cells (ILC2s) compared to uninfected mice (% ILC2: 10.28 ± 2.283 (infected) vs. 2.075 ± 0.340 (uninfected), P<0.05, n=4-6/group). Histological analysis of colitis showed a trend of higher scores in infected mice compared to uninfected mice (6.250 ± 1.153 (infected) vs. 4.750 ± 1.785 (uninfected), P=0.4905, n=4-6/group). Conclusion: Our data demonstrate that C. tropicalis may play a pro-inflammatory role in intestinal injury by exacerbating gut inflammation during the recovery phase of DSS-induced colitis. We speculate that infection with the fungus C. tropicalis may play a role in triggering flares during CD and that anti-fungal therapy may be beneficial in CD patients.
Background: Hypermutant malignancies in adults have shown a positive response to immune checkpoint inhibitor therapy. Patients with biallelic mismatch repair deficiency syndrome (BMMRD) have a unique cancer phenotype with hypermutation in the tumors. We postulated that young patients with inoperable hypermutated cancers respond to immune checkpoint inhibition therapy. Methods: Patients referred to the International Biallelic Mismatch Repair Deficiency Consortium were confirmed to have BMMRD, Lynch syndrome, POLE or POLD1 germline mutations based on the identification of germline mutations and/or immunohistochemical confirmation. Exome or whole genome sequencing were performed on available tumors. Immune check point inhibitor therapy (ICI) were offered to patients with hypermutant inoperable malignancies. Results: Eighty-one patients were identified with 119 tumors. Malignancies include brain (48%), colorectal (28%), hematological (16%), genitourinary (2%) and other (n=6%). Oligo-adenomatous colorectal polyposis occurred in 79% of patients screened. Classification of patients include BMMRD (n=69), Lynch syndrome (n=7), POLE (n=3) and under evaluation (n=2). Among the 69 BMMRD patients, PMS2 mutations were most frequent (n=42), followed by MSH6 (n=16), MSH2 (n=5), MLH1 (n=4), and EPCAM (n=2). The Lynch syndrome patients had MSH2 (n=4), MLH1 (n=2), and unknown (n=1) mutations. BMMRD brain tumors had the highest mutation loads (mean 17 740+/-7703 mutations per exome) mostly due to secondary polymerase mutations. BMMRD colorectal tumors were hypermutant (mean 1589+/-1043 mutations per exome). Twenty patients were treated with ICI therapy: 16 BMMRD and 4 Lynch syndrome. Mean age at primary cancer diagnosis was 11.6 years (SD +/- 7, range 1.8 to 27.4 years). Three patients had double primary cancer in different organs, two of which had distant metastatic disease. All 4 colorectal, the one genitourinary and 4 of 9 brain cancers had measureable clinical and radiological response to therapy with an overall survival at one year of 61%. Conclusion: Cancers in patients with BMMRD, POLE mutations and some Lynch cancers have a hypermutant tumor phenotype. These data are promising, particularly the response of the colorectal and genitourinary tumors to therapy. A large international clinical trial for management of hypermutant cancers has recently commenced and will provide further validation for these novel observations.
S-141
AGA Abstracts
AGA Abstracts
648