Transport Of L-glutamine Research Articles

Abstract P038: Early phase II clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) in adolescents and young adults (AYA) with brain cancers

Abstract Background: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a poly-chlorinated pyridine cholesteryl carbonate with a MOA via bis-alkylation of DNA @ N7-guanine and N4-cytosine that is currently in Phase II trial in AYA subjects with cancer that have CNS involvement. The aims for the trials are to assess clinical responses, monitor toxicities, safety and confirm the MTDs for IV administered DM-CHOC-PEN (IND 68,876) to AYA subjects with cancer involving the CNS. We report here responses and toxicities observed to date in Phases I & II DM-CHOC-PEN clinical trials with AYA subjects that had cancer involving the CNS. Subjects & Methods: DM-CHOC-PEN was administered as a 3-hr IV infusion once every 21 days to AYA subjects. The dosing schedule was 2-tiered: subjects with liver involvement received 85.8 mg/m2 and subjects with normal liver function received 98.7 mg/m2. Results: nineteen (19) AYA subjects with CNS involvement have been treated to date. The common tumor types treated were oligoastrocytoma, astrocytoma, GBM; leukemia (ALL), lymphoma (NHL), melanoma, breast and lung cancers (NSCL). Three (3) AYA patients are currently being followed with diagnoses of breast cancer, astrocytoma, and lung cancer and have had good qualities of life at 12, 59 and 72+ mos. All patients have been followed with lab tests, scans (RECIST 1.1) and virtual exams. The drug was well tolerated. The most common adverse effect was fatigue (17%). No neuro/cognitive, liver dysfunction, hematological, cardiac, renal or GI toxicities were observed. PK modeling revealed that AUCs were parallel for all dose levels. The Cmax for DM-CHOC-PEN and DM-PEN (4-demethylpenclomedine, a metabolite) were 3 and 24 hours, respectively for the AYA subjects; similar to what was seen for older adults. The drug and metabolite were still detectable in plasma and rbcs for 21 to 50 days in the AYA (15 – 39 y/o) group vs. 3 to < 21 days (previously reported for adult subjects (>60 y/o) (AACR #1185, 2013). Differences in PK profiles between AYA and older adult subjects will also be reviewed in depth. Conclusion: DM-CHOC-PEN is safe for usage and has produced objective responses with manageable toxicities in AYA subjects with cancer involving the CNS. Complete data on subject responses and observed toxicities will be presented. We propose a 3-stage mechanism for drug entry into the CNS and into cancer cells via reversible binding with RBCs and then association with L-glutamine transport into cells. Supported by NCI/SBIR grants – R43/44CA132257 and R43CA203351 and NIH NIGMS 1 U54 GM104940. Citation Format: Lee Roy Morgan, Roy S. Weiner, Tallat Mahmood, Marcus L. Ware, Andrew H. Rodgers, Manish Bhandari, Philip Friendlander. Early phase II clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) in adolescents and young adults (AYA) with brain cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P038.

Abstract CT152: Phase II clinical trial results for 4-demethyl-4-cholesteryloxycarbonyl-penclomedine (DM-CHOC-PEN) in advanced non-small cell lung cancer (NSCLC) involving the CNS

Abstract Background: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a poly-chlorinated pyridine cholesteryl carbonate with a MOA via bis-alkylation of DNA @ N7-guanine and N4-cytosine that has completed Phase I and II trials (the latter including subjects with CNS involvement) [AACR #1185, 2013; AACR #CT 129, 2019]. The primary aims of the previously reported Phase I/II DM-CHOC-PEN trials were to assess clinical response and monitor toxicities/safety and verify the maximum tolerated doses (MTD) for IV administered DM-CHOC-PEN (IND 68,876) to subjects with cancer. We report here the responses and toxicities seen in subjects with NSCLC involving the CNS that lacked genetic rearrangements, tumor targets and/or had failed standard therapies. Subjects & Methods: DM-CHOC-PEN was administered to adults (> 18 y/o) as a 3-hr IV infusion once every 21 days employing a verified 2-tiered MTD schedule: 85.8 mg/m2 for subjects with liver involvement and 98.7 mg/m2 for subjects with normal livers. Results: Fifty two (52) adult subjects with cancer have been treated to date, including16 subjects with lung cancer, of which 11 had NSCLC (adeno/large cell carcinomas) involving the CNS that lacked genetic rearrangements/no tumor targets and/or had failed standard therapies. Seven of the 11 subjects with NSCLC involving the CNS also possessed cerebellar metastases. The drug was well tolerated; the most common adverse effects were fatigue (17%), reversible liver dysfunction (9%) and nausea (11%). No neuro/psychological, hematological, cardiac or renal toxicities were observed, nor drug associated deaths. PK modeling revealed that AUCs were parallel for both the 85.8 and 98.7 mg/m2 doses employed. The Cmax for DM-CHOC-PEN and DM-PEN (4-demethylpenclomedine, a metabolite) at the MTD were 3 and 24 hours, respectively. Both DM-CHOC-PEN and DM-PEN were detected for up to 15 days post administration associated with rbc's. DM-CHOC-PEN was also detected in CNS tumor tissue obtained anatomically from five (5) subjects - in concentrations of 75-210 ng/g, 22 days to 9 mos. post-treatments. 8 subjects have responded with CR/PR (RECIST 1.1) and improved OS/QOL/PFS (Kaplan-Meier) lasting 8+ - 70+ mos. Conclusion: DM-CHOC-PEN is a bis-alkylator of DNA that is safe at the dose levels described and has produced long term objective responses with manageable toxicities in subjects with NSCLC involving the CNS lacking genetic rearrangements or tumor targets and/or had failed standard therapies. Complete data on subject responses and observed toxicities will be presented. A 3-stage mechanism is proposed for drug entry into the CNS and into NSCLC cells via reversible binding with RBC's and then associated with L-glutamine transport into cells. Supported by NCI/SBIR grants - R43/44CA132257 and NIH NIGMS 1 U54 GM104940 - the latter funds from the Louisiana Clinical and Translational Science Center, New Orleans, LA. Citation Format: Roy S. Weiner, Lee Roy Morgan, Marcus Ware, Tallat Mahmood, Craig Gordon, Manish Bhandari, Andrew Rodgers, Marc Matrana, Thomas M. Cosgriff, Philip Friedlander, Jay J. Zou. Phase II clinical trial results for 4-demethyl-4-cholesteryloxycarbonyl-penclomedine (DM-CHOC-PEN) in advanced non-small cell lung cancer (NSCLC) involving the CNS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT152.

Abstract CT181: Phase I/II clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) as treatment for adolescents and young adults (AYA) with cancers involving the CNS

Abstract Background: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a poly-chlorinated pyridine cholesteryl carbonate with a MOA via bis-alkylation of DNA @ N7-guanine and N4-cytosine that has completed Phase I and now in Phase II clinical trials in AYA subjects with cancer with CNS involvement. The aims were to assess clinical responses, monitor toxicities/safety and confirm MTDs for IV administered DM-CHOC-PEN in AYA individuals (IND 68,876). We report here responses, pharmacokinetics and toxicities seen in Phase I/II DM-CHOC-PEN trials with AYA subjects +/- CNS involvement. Subjects & Methods: DM-CHOC-PEN was administered as a 3-hr IV infusion once every 21 days to subjects with advanced cancer in escalating doses from 50 to 98.7 mg/m2. The MTD identified was similar to that seen during the adult Phase I trial – 85.8 mg/m2 for subjects with liver involvement and 98.7 mg/m2 for subjects with normal liver function. Results: Fifteen (15) AYA subjects have been treated to date (with or without CNS involvement). The common tumor types treated were oligoastrocytoma, astrocytoma, GBM; leukemia (ALL), lymphoma (NHL), melanoma, breast and lung cancers (NSCL). Most subjects (12) had CNS involvement. The drug was well tolerated; the most common adverse effects were fatigue (17%). No neuro/cognitive, liver dysfunction, hematological, cardiac, renal or GI toxicities were observed. PK modeling revealed that AUCs were parallel for all dose levels (50-98.7 mg/m2). The Cmax for DM-CHOC-PEN was 100 hours for the AYA subjects; very different to what has been observed for older adults (>50 y/o). The drug was also cleared quicker, as compared to older adults (2o to less existing co-morbidities in the AYA subjects). However, the drug was detectable in plasma and rbcs for 15 days in the AYA (15 – 39 y/o). Differences in PK profiles between AYA and older adult subjects will be reviewed in depth. Three (3) of the AYA subjects (1 each with NSCLC, astrocytoma and breast cancers involving the CNS) have responded with CR/PR (RECIST 1.1), improved QOL/PFS (Kaplan-Meier) and OS from 12 to 58+ mos. All subjects will be reviewed. Conclusion: DM-CHOC-PEN is safe in doses of 39 – 98.7 mg/m2 and has produced objective responses with manageable toxicities in AYA subjects with cancer involving the CNS. Complete data on subject responses and observed toxicities will be presented. We propose a 3-stage mechanism for drug entry into the CNS and into cancer cells via reversible binding with RBCs and then association with L-glutamine transport into cells. Supported by NCI/SBIR & LA state grants – R43/44CA132257, R43/44CA203351 and NIH NIGMS 1 U54 GM104940 – the latter funds the Louisiana Clinical and Translational Science Center. Citation Format: Lee Roy Morgan, Roy S. Weiner, Tallat Mahmood, Marcus Ware, Andrew H. Rodgers, Manish Bhandari, Philip Friedlander. Phase I/II clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) as treatment for adolescents and young adults (AYA) with cancers involving the CNS [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT181.

Glutamine-induced signaling pathways via amino acid receptors in enteroendocrine L cell lines.

Glucagon-like peptide-1 (GLP-1), secreted by gastrointestinal enteroendocrine L cells, induces insulin secretion and is important for glucose homeostasis. GLP-1 secretion is induced by various luminal nutrients, including amino acids. Intracellular Ca2+ and cAMP dynamics play an important role in GLP-1 secretion regulation; however, several aspects of the underlying mechanism of amino acid-induced GLP-1 secretion are not well characterized. We investigated the mechanisms underlying the L-glutamine-induced increase in Ca2+ and cAMP intracellular concentrations ([Ca2+]i and [cAMP]i, respectively) in murine enteroendocrine L cell line GLUTag cells. Application of L-glutamine to cells under low extracellular [Na+] conditions, which inhibited the function of the sodium-coupled L-glutamine transporter, did not induce an increase in [Ca2+]i. Application of G protein-coupled receptor family C group 6 member A and calcium-sensing receptor antagonist showed little effect on [Ca2+]i and [cAMP]i; however, taste receptor type 1 member 3 (TAS1R3) antagonist suppressed the increase in [cAMP]i. To elucidate the function of TAS1R3, which forms a heterodimeric umami receptor with taste receptor type 1 member 1 (TAS1R1), we generated TAS1R1 and TAS1R3 mutant GLUTag cells using the CRISPR/Cas9 system. TAS1R1 mutant GLUTag cells exhibited L-glutamine-induced increase in [cAMP]i, whereas some TAS1R3 mutant GLUTag cells did not exhibit L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. These findings suggest that TAS1R3 is important for L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. Thus, TAS1R3 may be coupled with Gs and related to cAMP regulation.

Inhibition of cell proliferation by L-theanine transported into cells via an L-glutamine transporter Slc38a1

Inhibition of cell proliferation by L-theanine transported into cells via an L-glutamine transporter Slc38a1

Protein kinase C-mediated impairment of glutamine outward transport and SN1 transporter distribution by ammonia in mouse cortical astrocytes

SN1, a system N amino acid transporter specific for astrocytes, is mainly responsible for export of newly synthesized L-glutamine from the cells. Astrocytic retention of L-glutamine which plays a critical role in ammonia-induced astrocytic swelling resulting in brain edema, could be tentatively attributed to the impaired L-glutamine export from astrocytes. The present study demonstrates that treatment of cultured mouse cortical astrocytes for 24 h with 5 mM ammonium chloride (“ammonia”) inhibits the system N-mediated L-glutamine transport out of the cell, and that this inhibition is related to the reduced presence of the SN1 transporter on the cell membrane. Ammonia decreased total protein kinase C (PKC) activity in the absence but not in the presence of PKC activator, phorbol 12-myristate 13-acetate (PMA), and activation of PKC by PMA reversed both the ammonia-induced decrease of system N-mediated L-glutamine release and ammonia-induced SN1 deficit in the membrane fraction. However, while ammonia did not change the protein level of PKCα isoform, it decreased the protein content of PKCδ. Moreover, ammonia treatment increased the cell surface expression of SN1 in cells with silenced PKCα and PKCδ. Silencing of PKCδ abrogated the decrease of system N (SN1)-mediated L-glutamine release by ammonia. The results implicate the involvement of PKCδ in the inhibition of SN1 membrane expression and activity by ammonia.

Abstract A086: Early clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) in adolescents and young adults (AYA) with cancers

Abstract Background: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a poly-chlorinated pyridine cholesteryl carbonate with a MOA via bis-alkylation of DNA @ N7-guanine and N4-cytosine that has completed phase I and II trials in adult subjects with cancer that had +/- CNS involvement (AACR #1185, 2013; AACR #CT 129, 2017). The aims were to assess clinical responses, monitor toxicities/safety, and confirm MTDs for IV administered DM-CHOC-PEN (IND 68,876). We report here responses and toxicities seen in a phase I DM-CHOC-PEN trial with AYA subjects who have cancer (some of whom had CNS involvement). Subjects and Methods: DM-CHOC-PEN was administered as a 3-hr IV infusion once every 21 days to subjects with advanced cancer in escalating doses from 39 - 98.7 mg/m2. The dosing schedule accounted for the liver toxicities seen in the adult phase I trial and was 2-tiered: for subjects with liver involvement, the cutoff was 85.8 mg/m2 and for subjects with normal liver function was 98.7 mg/m2. Results: Twelve (12) AYA subjects have been treated to date (with or without CNS involvement). The common tumor types treated were oligoastrocytoma, astrocytoma, GBM; leukemia (ALL), lymphoma (NHL), melanoma, breast, and lung cancers (NSCL). Most subjects (9) had CNS involvement. The drug was well tolerated; the most common adverse effects were fatigue (17%). No neuro/cognitive, liver dysfunction, hematologic, cardiac, renal, or GI toxicities were observed. PK modeling revealed that AUCs were parallel for all dose levels (39-98.7 mg/m2). The Cmax for DM-CHOC-PEN and DM-PEN (4-demethylpenclomedine, a metabolite) were 3 and 24 hours, respectively for the AYA subjects, similar to what was seen for older adults. However, the drug and metabolite were still detectable in plasma and rbcs for 21 to 50 days in the AYA (15 - 39 yo) group vs. 3 to < 21 days (as observed in previously treated adult (>60 yo) subjects (AACR #1185, 2013). Differences in PK profiles between AYA and older adult subjects will be reviewed in depth. Three (3) of the AYA subjects (1 each with NSCLC, ALL, and astrocytoma involving the CNS) have responded with CR/PR (RECIST 1.1), improved QOL/PFS (Kaplan-Meier), and OS from 8 to 26+ mos. All subjects will be reviewed. Conclusion: DM-CHOC-PEN is safe in doses of 39 - 98.7 mg/m2 and has produced objective responses with manageable toxicities in AYA subjects with cancer involving the CNS. Complete data on subject responses and observed toxicities will be presented. We propose a 3-stage mechanism for drug entry into the CNS and into cancer cells via reversible binding with RBCs and then association with L-glutamine transport into cells. Supported by NCI/SBIR grants - R43/44CA132257 and R43CA203351A and NIH NIGMS 1 U54 GM104940. Citation Format: Lee Roy Morgan, Andrew H. Rodgers, Roy S. Weiner, Tallat Mahmood, Marcus L. Ware, Manish Bhandari, Philip Friedlander. Early clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) in adolescents and young adults (AYA) with cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A086.

Abstract CT052: Clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) in cancers involving the CNS

Abstract Background: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a poly-chlorinated pyridine cholesteryl carbonate with a MOA via bis-alkylation of DNA @ N7-guanine and N4-cytosine that has completed Phase I and II trials (the latter on subjects with CNS involvement) [AACR #1185, 2013; AACR #CT 129]. The primary aim was to assess clinical response and secondary aims to monitor toxicities/safety and verify the MTDs for IV administered DM-CHOC-PEN that derived in Phase I study (IND 68,876). We report here the responses and toxicities seen in all the subjects treated. Subjects & Methods: In Phase I, DM-CHOC-PEN was administered as a 3-hr IV infusion once every 21 days to subjects with advanced cancer; cohorts received escalating doses from 39 - 111 mg/m2. The Phase II dose schedule was 2-tiered: 85.8 mg/m2 for subjects with liver involvement and 98.7 mg/m2 for subjects with normal livers. Results: Fifty two (52) subjects have been treated to date - 25 in Phase I (cancer subjects with or without CNS involvement) and 27 in Phase II (with CNS involvement). The common tumor types treated were primary brain cancers and melanoma, breast, and lung cancers involving the CNS. The drug was well tolerated; the most common adverse effects were fatigue (17%), reversible liver dysfunction (9%) and nausea (11%). No neuro/psychological, hematological, cardiac or renal toxicities were observed. PK modeling revealed that AUCs were parallel for all dose levels (39-111 mg/m2). The Cmax for DM-CHOC-PEN and DM-PEN (4-demethylpenclomedine, a metabolite) were 3 and 24 hours, respectively. Both DM-CHOC-PEN and DM-PEN were detected 3 to 15 days after administration associated (up to 50%) with rbcs. Of interest, young adults (<40 y/o) demonstrated significant increases in Cmax and AUC vs. older subjects, supporting the need for trials in adolescents and young adults. DM-CHOC-PEN was also detected in CNS tumor tissue obtained surgically from five (5) subjects - in concentrations of 75-210 ng/g, 22 days to 9 mos. post treatments at doses of 39 or 98.7 mg/m2 of drug. To date, 16 subjects with lung cancer (11 with NSCLC involving the CNS) have been treated. Seven of the 11 subjects with NSCLC involving the CNS (incl. 6 with cerebellar disease) have responded with CR/PR (RECIST 1.1) and improved OS/QOL/PFS (Kaplan-Meier) lasting 8+ - 32+ mos. Conclusion: DM-CHOC-PEN is safe at these dose levels and has produced objective responses with manageable toxicities in subjects with cancer involving the CNS. Complete data on subject responses and observed toxicities will be presented. We propose a 3-stage mechanism for drug entry into the CNS and into NSCLC cells via reversible binding with RBCs and then associated with L-glutamine transport into cells. Supported by NCI/SBIR grants - R43/44CA132257 and NIH NIGMS 1 U54 GM104940 - the latter funds the Louisiana Clinical and Translational Science Center. Citation Format: Roy S. Weiner, Lee Roy Morgan, T Mahmood, R. Kawauchi, C. Gordon, ML Ware, M. Matrana, TM Cosgriff, AH Rodgers, G. Bastian, M. Bhandari, J-J Zou. Clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) in cancers involving the CNS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT052. doi:10.1158/1538-7445.AM2017-CT052

Amino acid transporter SLC38A3 promotes metastasis of non-small cell lung cancer cells by activating PDK1

BackgroundTumor metastasis is a finely-tuned pathological process coupled to metabolic reprogramming that includes both glutamine and glucose. The solute carrier SLC38A3, a member of amino acid/polyamine/organocation (APC) superfamily, is an l-glutamine transporter. It is not clear whether SLC38A3 involves the metastasis of NSCLC (non small cell lung cancer). MethodsThe scratch test and transwell assay were used to determine the ability of NSCLC to migrate. Cellular amino acids content was determined by mass spectrometry. The cellular response to glutamine/histidine deficiency was evaluated in A549 cells. The expression of SLC38A3 was assayed in clinical NSCLC and paratumor tissues by histoimmunochemistry staining. A nude mouse model of NSCLC metastasis was developed by tail vein injection of tumor cells. ResultsSLC38A3 was upregulated in metastatic NSCLC cells and its expression was correlated with prognosis of NSCLC patients. SLC38A3 overexpression promoted epithelial – mesenchymal transition (EMT) and migration of HCC827 and A549 human lung adenocarcinoma cells, and accelerated tumor metastasis in mice. We found that SLC38A3 decreased the cellular concentrations of glutamine and histidine, and the deficiency of glutamine or histidine activated PDK1/AKT signaling that in turn, triggered NSCLC metastasis. ConclusionsSLC38A3 activated PDK1/AKT signaling and promoted metastasis of NSCLC through regulating glutamine and histidine transport, suggesting SLC38A3 as a potential therapeutic target for treatment of NSCLC.

N-Glycosylation influences transport, but not cellular trafficking, of a neuronal amino acid transporter SNAT1.

SNAT1 is a system N/A neutral amino acid transporter that primarily expresses in neurons and mediates the transport of l-glutamine (Gln). Gln is an important amino acid involved in multiple cellular functions and also is a precursor for neurotransmitters, glutamate and GABA. In the present study, we demonstrated that SNAT1 is an N-glycoprotein expressed in neurons. We identified three glycosylation sites at asparagine residues 251, 257 and 310 in SNAT1 protein, and that the first two are the primary sites. The biotinylation and confocal immunofluorescence analysis showed that the glycosylation-impaired mutants and deglycosylated SNAT1 were equally capable of expressing on the cell surface. However, l-Gln and 3H-labeled methyl amino isobutyrate (MeAIB) was significantly compromised in N-glycosylation-impaired mutants and deglycosylated SNAT1 when compared with the wild-type control. Taken together, these results suggest that SNAT1 is an N-glycosylated protein with three de novo glycosylation sites and N-glycosylation of SNAT1 may play an important role in the transport of substrates across the cell membrane.

Abstract CT129: Phase II clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) in NSCLC involving the CNS

Abstract Background: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a poly-chlorinated pyridine cholesteryl carbonate with a MOA via bis-alkylation of DNA @ N7-guanine and N4-cytosine that has completed a Phase I study [AACR #1185, 2013] and is being evaluated in a Phase II trial in patients with primary brain cancers and with melanoma, breast, and lung cancers with metastases to brain. The aims are to assess clinical response when DM-CHOC-PEN is administered I.V. at MTD and to monitor duration of responses and safety (IND 68,876). We report here the responses and toxicities seen in patients with NSCLC involving the CNS. Patients & Methods: In Phase I, DM-CHOC-PEN was administered as a 3-hr IV infusion once every 21 days to patients with advanced cancer; cohorts received escalating doses from 39 - 111 mg/m2. The Phase II dose schedule is 2-tiered: 85.8 mg/m2 for patients with liver involvement and 98.7 mg/m2 for patients with normal livers. Results: Fifty two (52) patients have been treated to date - 26 in Phase I (cancer patients with or without CNS involvement) and 26 in Phase II (with CNS involvement). The drug was well tolerated; the most common adverse effects were fatigue (17%), reversible liver dysfunction (9%) and nausea (11%). No neuro/psychological, hematological, cardiac or renal toxicities were observed. PK modeling revealed that AUCs were parallel for all dose levels (39-111 mg/m2). The Cmax for DM-CHOC-PEN and DM-PEN (4-demethylpenclomedine, a metabolite) were 3 and 24 hours, respectively. Both DM-CHOC-PEN and DM-PEN were detected 3 to 15 days after administration associated (up to 50%) with rbcs. DM-CHOC-PEN was also detected in CNS tumor tissue obtained surgically from five (5) patients - concentrations of 75-210 ng/g, 22 days to 9 mos. post treatments at doses of 39 or 98.7 mg/m2 of drug. To date, 16 patients with lung cancer (11 with NSCLC involving the CNS) have been treated. Seven of the 11 patients with NSCLC involving the CNS (incl. 6 with cerebellar disease) have responded with CR/PR (RECIST 1.1) and improved OS/QOL/PFS (Kaplan-Meier) lasting 6+ - 21+ mos. Conclusion: DM-CHOC-PEN is safe at these dose levels and has produced objective responses with manageable toxicities in NSCLC involving the CNS. Complete data on patient responses and observed toxicities will be presented. We propose a 2-stage mechanism for drug entry into the CNS and into NSCLC cells via reversible binding with RBCs and then L-glutamine transport into cells. Supported by NCI/SBIR grants - R43/44CA132257 and NIH NIGMS 1 U54 GM104940 - the latter funds the Louisiana Clinical and Translational Science Center. Citation Format: Roy S. Weiner, T Mahmood, C Gordon, ML Ware, LR Morgan, TM Cosgriff, AH Rodgers, G Bastian, R Kawauchi, M Matrana, J-J Zou. Phase II clinical trial results for 4-demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) in NSCLC involving the CNS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT129.

Abstract C152: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) as a cytotoxic L-glutamine agonist in non-small cell lung cancer (NSCLC) involving the CNS

Abstract Purpose: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a poly-chlorinated pyridine cholesteryl carbonate whose MOA is via bis-alkylation of DNA @ N7 - guanine and N6 - cytosine. DM-CHOC-PEN has undergone a Phase I study (allowed enrollment of subjects with advanced cancer +/- CNS involvement) and is being evaluated in a Phase II trial in subjects with advanced cancer involving the brain. Impressive objective responses and improved PFS/overall survival have been observed in subjects with NSCLC involving the CNS. The main aims of this presentation are to document a mechanism for DM-CHOC-PEN's penetration into NSCLC metastases involving the CNS via L-glutamine (GLM) transport. Methods: Human cancer cell lines [NSCLC - H2087 & H460 and SCLC - SHP-77 & H1417 - obtained from ATCC] were maintained in culture with complete RPMI (5% FBS, pen/strep/Amp. B), supplemented w/ L-glutamine [300 mg/500 mL medium, Sigma] @ 37oC and in moist 5% CO2 conditions. DM-CHOC-PEN was dissolved in tetrahydrofuran, impregnated into ChemoChads® that deliver drug 0.25 - 5 μg/mL in culture. Cells were grown in culture w/wo supplemental GLM for 36 h and then ChemoChads were added. Cytotoxicity was conducted in multi-well plates using the Cytotec® Assay and measuring IC50 values after 24 hr. All assays were conducted in triplicate. Results: All 4-cell lines grew well in complete RPMI supplemented with L-glutamine. Both SCLC cell lines grew well as clumps of small single cells in suspension, +/- GLM suppl. but were not sensitive to DM-CHOC-PEN, w/ or wo/ GLM supplements. In contrast, both NSCLC cell lines grew as coalescing islands of adhered well-differentiated cells in GLM supplemented medium, but did not grow well in GLM-free medium. Of interest, both NSCLC cell lines were sensitive to DM-CHOC-PEN (IC50 0.25-0.75 μg/mL) w/ GLM-suppl. medium, but were not sensitive (IC50 >5 μg/mL) in GLM-free medium. However, when GLM was added to the GLM-free medium w/ DM-CHOC-PEN, the cells resumed sensitivity to the drug. Similar findings have been observed for 6/7-human NSCLC explants obtained from CNS surgical samples (which will be discussed). Conclusion: The common structural similarities between DM-CHOC-PEN, GLM and Ɣ-aminobutyric acid (GABA) could allow the sharing of a transport/receptor mechanism into the cerebellum and other CNS safe havens that provide a favorable microenvironment for NSCLC cells to colonize, thrive and grow. The drug when administered IV associates with erythrocytes (∼50%), which facilitates its entry into the neoplastic cerebral circulation and now support for its transport into metastatic NSCLC involving the CNS via the GLM transport system is presented. In the absence of GLM, the GLM-transport system shuts down. Of clinical interest is that all subjects with cerebellar NSCLC lesions have responded to DM-CHOC-PEN in the clinical trials with objective responses, improved PFS (6-17+ months) and improved QOL (AACR, Abst. 1161, 2015). The cerebellum offers a unique microenvironment rich in chemical transmitters and amino acids - in particular GLM and Ɣ-aminobutyric acid (GABA). Supported by NCI/SBIR grant - R43/44CA132257. Citation Format: Lee Roy Morgan, Jr., Ed Benes, Andrew H. Rodgers, Tallat Mahmood, Roy S. Weiner, Marcus L. Ware. 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) as a cytotoxic L-glutamine agonist in non-small cell lung cancer (NSCLC) involving the CNS. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C152.

The regulation and role of L-glutamine in B-lymphocyte activation (LYM7P.618)

Abstract B lymphocyte activation is a highly energetic event that promotes cell survival and growth. Aberrant activation of B lymphocytes can lead to disease states such as lymphomas or autoimmune diseases. To meet the metabolic demands of activation, B cells increase nutrient uptake to provide energy and intermediates for macromolecule synthesis. We have investigated the role of L-glutamine, which is emerging as a critical immunomodulatory nutrient; however, the precise role of L-glutamine in B cell growth responses is poorly understood. Depletion of extracellular L-glutamine results in impaired cell growth and proliferation in response to B-cell antigen receptor (BCR) ligation, although, early activation events such as induction of CD86 are unaffected. We show that L-glutamine uptake is induced in response to BCR ligation. This corresponds to an increase in the surface expression of the high affinity L-glutamine transporter, ASCT2, indicating its involvement in L-glutamine uptake. Further, inhibition of ASCT2 by the small molecule N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) caused impaired B lymphocyte activation similar to that observed in response to L-glutamine depletion. The addition of L-leucine abated some of the effects of GPNA treatment on B lymphocyte growth responses, indicating mTORC involvement. These data provide new insight on the requirement of L-glutamine in B lymphocyte activation and suggest a possible underlying mechanism of growth regulation by mTORC.

<i>In Vitro</i> Study of L-Glutamate and L-Glutamine Transport in Retinal Pericytes: Involvement of Excitatory Amino Acid Transporter 1 and Alanine–Serine–Cysteine Transporter 2

L-Glutamate (L-Glu) is known to be a relaxant of pericytes and to induce changes in microcirculatory hemodynamics. Since the concentration of L-Glu which induces the dilation of retinal capillaries is reported to be high compared with the estimated concentration in the retinal interstitial fluid, it is hypothesized that some systems involving concentrative L-Glu release are present in retinal pericytes. The purpose of this study was to investigate the existence of L-Glu-storing systems, which contribute to autocrine L-Glu release, in retinal pericytes using conditionally immortalized rat retinal pericytes (TR-rPCT1 cells), which express mRNAs of L-Glu-synthesizing enzymes from L-glutamine (L-Gln). TR-rPCT1 cells express the mRNAs of vesicular L-Glu transporter 1 (VGLUT1), indicating that L-Glu in the cytoplasm is taken up into VGLUT1-expressing vesicles of retinal pericytes. L-Glu and L-Gln are taken up into TR-rPCT1 cells via Na(+)-dependent saturable process(es) with a Km value of 22.4 µM and 163 µM, respectively. The [(3)H]L-Glu uptake was inhibited by ca. 50% in the presence of D-aspartate, a substrate of excitatory amino acid transporter (EAAT) subtypes, whereas substrates of alanine-serine-cysteine transporter (ASCT) subtypes exhibited only a weak inhibitory effect on [(3)H]L-Glu uptake compared with D-aspartate. Regarding the L-Gln uptake by TR-rPCT1 cells, the inhibitory effect of ASCT substrates on the [(3)H]L-Gln uptake was stronger than that of substrates of other neutral amino acid transport systems. Consequently, it was determined that EAAT1 and ASCT2 play a role in the transport of L-Glu and L-Gln, respectively, from retinal interstitial fluid to the cytoplasm of retinal pericytes.

Abstract 2440: RNF5 mediates ER stress-induced degradation of SLC1A5 in breast cancer

Abstract Tumor cells have an altered metabolism that distinguishes it from their normal counterpart. Intriguingly, tumor cells show increased use of glutamine to fuel catabolic processes. Therefore, altered metabolic tumors have been considered as a good target for anticancer therapy. Ring finger protein 5 (RNF5) is an ER membrane-associated ubiquitin E3 ligase. RNF5 expression has been associated with breast cancer progression. We identified solute carrier family 1 member 5 (SLC1A5) as a possible RNF5 regulated protein using a MS analysis of breast cancer cells. SLC1A5 is a high affinity L-glutamine transporter, which is highly expressed in tumors such as breast, liver and lung. RNF5 promotes ubiquitination-mediated degradation of SLC1A5 in response to chemotherapeutic drugs known to induce ER stress. RNF5-mediated SLC1A5 instability attenuated mTOR signaling, leading to inhibition of tumor cell proliferation and promotion of apoptosis/autophagy. The loss of RNF5 in PyMT-MMTV crossed with RNF5 mutant animals developed less differentiated mammary tumors, which exhibit increased levels of SLC1A5. TMA analysis of human breast cancer tumors identified a fraction that exhibited an inverse correlation between RNF5 and SLC1A5 expression. Notably, reduced expression of SLC1A5 exhibited a strong association with improved prognosis of breast cancer. Taken together, our observations identify mechanisms underlying the control of SLC1A5 stability, and the implications of such regulation for mTOR signaling and breast cancer survival in response to therapy. Citation Format: Young Joo Jeon, Sihem Khelifa, Yongmei Feng, Eric Lau, Robert Cardiff, Hyungsoo Kim, David L. Rimm, Yuval Kluger, Ze'ev Ronai. RNF5 mediates ER stress-induced degradation of SLC1A5 in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2440. doi:10.1158/1538-7445.AM2014-2440

Cello-oligosaccharide influences intestinal microflora, mucosal architecture and nutrient transport in weaned pigs

Effects of cello-oligosaccharide (COS) on growth performance, intestinal microflora, mucosal architecture and nutrient transport of weaned pigs were investigated. A total of 144 piglets, with an average weight of 6.13±0.59kg weaned at 21±1d age, were randomly allotted to four groups for two weeks. The four treatments were the control (basal diet), and the basal diet supplemented with 1.5, 3.0, 4.5g/kg COS, respectively. The results showed that COS supplementation had no effect (P>0.05) on average daily feed intake, daily gain and gain/feed ratio. Incremental levels of COS increased Lactobacillus (linear P=0.003; quadratic P=0.007) and decreased Clostridium (linear P=0.005; quadratic P=0.007) in jejunal contents, increased villus height (linear P=0.005; quadratic P=0.007), villus height/crypt depth ratio (linear P=0.000; quadratic P=0.001) and villus surface area (linear P=0.009; quadratic P=0.012), increased RNA/DNA and protein/DNA ratio in jejunal mucosa linearly (P=0.006 and P=0.006) and quadratically (P=0.017 and P=0.009), respectively. The electrogenic nutrient transport from the jejunal mucosa in Ussing chambers showed that increasing COS level increased sodium-dependent glucose transport (linear P=0.009; quadratic P=0.016) and l-glutamine transport (linear P=0.029; quadratic P=0.080), respectively. The results indicated that COS has a positive effect on intestinal microflora, mucosal architecture and nutrient transport in the piglet small intestine after weaning, and the dietary COS offers a promising approach to alleviative post-weaning intestinal tract disorders in pigs.

L-glutamine transport and metabolism in the exocrine pancreas during acute pancreatitis

L-glutamine transport and metabolism in the exocrine pancreas during acute pancreatitis

Membrane Topological Structure of Neutral System N/A Amino Acid Transporter 4 (SNAT4) Protein

Members of system N/A amino acid transporter (SNAT) family mediate transport of neutral amino acids, including l-alanine, l-glutamine, and l-histidine, across the plasma membrane and are involved in a variety of cellular functions. By using chemical labeling, glycosylation, immunofluorescence combined with molecular modeling approaches, we resolved the membrane topological structure of SNAT4, a transporter expressed predominantly in liver. To analyze the orientation using the chemical labeling and biotinylation approach, the "Cys-null" mutant of SNAT4 was first generated by mutating all five endogenous cysteine residues. Based on predicted topological structures, a single cysteine residue was introduced individually into all possible nontransmembrane domains of the Cys-null mutant. The cells expressing these mutants were labeled with N-biotinylaminoethyl methanethiosulfonate, a membrane-impermeable cysteine-directed reagent. We mapped the orientations of N- and C-terminal domains. There are three extracellular loop domains, and among them, the second loop domain is the largest that spans from amino acid residue ∼242 to ∼335. The orientation of this domain was further confirmed by the identification of two N-glycosylated residues, Asn-260 and Asn-264. Together, we showed that SNAT4 contains 10 transmembrane domains with extracellular N and C termini and a large N-glycosylated, extracellular loop domain. This is the first report concerning membrane topological structure of mammalian SNAT transporters, which will provide important implications for our understanding of structure-function of the members in this amino acid transporter family.

W1578 ASCT2 and B°AT1 Are Involved in Leptin-Sensitive Na+-Dependent L-Glutamine Transport in Rat Small Intestine

W1578 ASCT2 and B°AT1 Are Involved in Leptin-Sensitive Na+-Dependent L-Glutamine Transport in Rat Small Intestine

Inhibition of SNAT2 by Metabolic Acidosis Enhances Proteolysis in Skeletal Muscle

Insulin resistance is a major cause of muscle wasting in patients with ESRD. Uremic metabolic acidosis impairs insulin signaling, which normally suppresses proteolysis. The low pH may inhibit the SNAT2 l-Glutamine (L-Gln) transporter, which controls protein synthesis via amino acid-dependent insulin signaling through mammalian target of rapamycin (mTOR). Whether SNAT2 also regulates signaling to pathways that control proteolysis is unknown. In this study, inhibition of SNAT2 with the selective competitive substrate methylaminoisobutyrate or metabolic acidosis (pH 7.1) depleted intracellular L-Gln and stimulated proteolysis in cultured L6 myotubes. At pH 7.1, inhibition of the proteasome led to greater depletion of L-Gln, indicating that amino acids liberated by proteolysis sustain L-Gln levels when SNAT2 is inhibited by acidosis. Acidosis shifted the dose-response curve for suppression of proteolysis by insulin to the right, confirming that acid increases proteolysis by inducing insulin resistance. Blocking mTOR or phosphatidylinositol-3-kinase (PI3K) increased proteolysis, indicating that both signaling pathways are involved in its regulation. When both mTOR and PI3K were inhibited, methylaminoisobutyrate or acidosis did not stimulate proteolysis further. Moreover, partial silencing of SNAT2 expression in myotubes and myoblasts with small interfering RNA stimulated proteolysis and impaired insulin signaling through PI3K. In conclusion, SNAT2 not only regulates mTOR but also regulates proteolysis through PI3K and provides a link among acidosis, insulin resistance, and protein wasting in skeletal muscle cells.