Philip Coffino - Academia.edu (original) (raw)
Papers by Philip Coffino
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Mar 12, 2013
PubMed, Nov 1, 1976
... SUMMARY INSEL, PAUL A., MAGUIRE, MICHAEL E., GILMAN, ALFRED G., BOURNE, HENRY R., C0FFIN0, PH... more ... SUMMARY INSEL, PAUL A., MAGUIRE, MICHAEL E., GILMAN, ALFRED G., BOURNE, HENRY R., C0FFIN0, PHILIP & MELMON, KENNETH L. (1976) Beta admenergic receptors and adenylate cyclase: products of separate genes? Mol. Pharmacol., 12, 1062-1069. ...
F1000 - Post-publication peer review of the biomedical literature, 2004
Science Advances, Jun 23, 2023
Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a... more Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a somatic dysregulation of protein kinase A. We show that the proteome of FLC tumors is distinct from that of adjacent nontransformed tissue. These changes can account for some of the cell biological and pathological alterations in FLC cells, including their drug sensitivity and glycolysis. Hyperammonemic encephalopathy is a recurrent problem in these patients, and established treatments based on the assumption of liver failure are unsuccessful. We show that many of the enzymes that produce ammonia are increased and those that consume ammonia are decreased. We also demonstrate that the metabolites of these enzymes change as expected. Thus, hyperammonemic encephalopathy in FLC may require alternative therapeutics.
Cancer Research, Jun 15, 2022
Fibrolamellar hepatocellular carcinoma (FLC) is a lethal pediatric cancer affecting adolescents a... more Fibrolamellar hepatocellular carcinoma (FLC) is a lethal pediatric cancer affecting adolescents and young adults. Besides surgical resection, there is no approved and effective therapy. FLC is driven by a fusion oncokinase, DNAJB1-PRKACA, that arises from a deletion fusing exon 1 of DNAJB1 and exons 2-10 of PRKACA, the catalytic subunit of protein kinase A. Expression of this chimeric oncokinase is capable of recapitulating the key histologic and transcriptomic features of FLC. Using PDXs, we tested therapeutics that have been used clinically for FLC or that target pathways identified via the transcriptomic signature of FLC, in addition to an agnostic library of clinical-stage drugs. Several compounds were identified with a varying degree of efficacy in different PDX lines as compared to our control line, primary human hepatocytes. One of the top hits was irinotecan, and its active metabolite SN38. Irinotecan is a topoisomerase I inhibitor and is clinically utilized in a wide variety of adult and pediatric cancers. A second top hit was Navitoclax, an inhibitor of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Tool compounds that targeted Bcl-xL, but not bcl-2 were efficacious. This suggested a possible link between Bcl-xL, which is overexpressed in some PDX lines, and the resistance profile they showed in our screen. Unfortunately, Navitoclax has an on-target and dose-limiting toxicity of thrombocytopenia, which limits its clinical application. DT2216 is a VHL-based PROTAC that degrades Bcl-xL. DT2216 has two potential benefits over Navitoclax: 1) DT2216 is a more potent than Navitoclax against various Bcl-xL dependent tumor cells; and 2) it is less toxic to platelets than Navitoclax by targeting Bcl-xl to VHL that is poorly expressed in platelets to avoid induction of severe thrombocytopenia. A phase 1 clinical trial of DT2216 in relapsed/refractory malignancies is now ongoing (NCT04886622). We tested if the combination of DT2216 and irinotecan might have a therapeutic potential in FLC. Indeed, in vitro screening of the proposed combo, DT2216 and SN38, showed an additive or a synergistical effect in 4 validated FLC cell lines. We tested both the efficacy of DT2216 on Bcl-xL levels in FLC as well as platelet counts to identify any developing thrombocytopenia. DT2216 was less disruptive to platelets than Navitoclax at therapeutically relevant pre-clinical doses. Next, we tested 4 of our PDX models for FLC using various dosing regimens to identify the most efficacious regimen with the minimum side effects. Mice cohorts were evaluated for weight loss and tumor volume. Mice treated with DT2216 and SN38 showed consistent therapeutic benefit which was not evident in controls and less notable as monotherapy. In conclusion, TOPO1 and Bcl-xL prove to be promising targets of interest in FLC. Targeting both with DT2216 and irinotecan leads to tumor shrinkage in pre-clinical models and offer a potential therapeutic/pharmacological intervention for FLC. Citation Format: Bassem Shebl, Gadi Lalazar, Denise Ng, Tova Finkelstein, Guangrong Zheng, Peiyi Zhang, Carly Rosemore, Michael Ortiz, Daohong Zhou, Sanford Simon, Philip Coffino. Targeting Bcl-xL in fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3888.
Cancer Research, Jun 15, 2022
Fibrolamellar hepatocellular carcinoma, FLC, is a usually lethal cancer affecting children, and y... more Fibrolamellar hepatocellular carcinoma, FLC, is a usually lethal cancer affecting children, and young adults. We have shown that all patients have a disruption in the ecology of protein kinase A activity. Hundreds of patients tested have a fusion of the first exon of DNABJ1, a heat shock protein cofactor, to PRKACA, the catalytic subunit of protein kinase A and expression of this DNAJB1-PRKACA fusion oncokinase is sufficient to produce the tumor. One patient is missing the regulatory subunit of protein kinase A and three patients have a fusion of the first exon of a different protein, ATP1B1, to the catalytic subunit of protein kinase A. We characterized the proteome and phosphoproteome of FLC cells relative to adjacent normal tissue. The changes were sufficiently characteristic to identify cell extracts from FLC, based solely on the proteome or phosphoproteome, and distinguishable from other tumor or normal liver. By calibrating protein level we found that tumor cells have an increase of catalytic subunit to such an extent as to exceed the capacity of protein kinase A regulatory subunits. This has two implications. First, there is free catalytic subunit that is unregulated, creating a high basal level of kinase activity. Second, the catalytic subunit is no longer localized by tethering to the regulatory subunit. Thus, the catalytic subunit has access to novel substrates. As an independent confirmation, we found the free basal kinase activity was higher in FLC cells. As a further test, we showed free catalytic subunit, that is not conjugated to regulatory subunit, is higher in FLC cells. We next tested whether these changes were solely due to overexpression of the catalytic subunit or if there was some additional change in the intrinsic activity of the kinase as a result of the fusion. We purified, without using an affinity tag, either PRKACA (which is myristoylated), DNAJB1-PRKACA, and PRKACA which was not myristoylated (the DNAJB1-PRKACA is not myristoylated). As a further control we also used the PRKACAL206R mutation, which does not engage regulatory subunits and is present in some forms of Cushing’s disease. We purified cytosol from human liver, blocked activity of all the endogenous kinases, and added our purified kinase. Mass spectrometry was then used to identify all newly phosphorylated proteins. While many proteins were equally phosphorylated by all four kinases (PRKACA, DNAJB1-PRKACA, PRKACA not myristoylated, PRKACAL206R) there were some distinct differences. From an analysis of these we found alterations in the optimal recognition sequence for phosphorylation for each variant of the kinase. Significantly, when we examined the human tissue data, we found many proteins that were phosphorylated by the DNAJB1-PRKACA in vitro were also phosphorylated in patient tumor tissue, but not in adjacent non-transformed tissue. This validated these observations of altered phosphorylation from the in vitro assays. Citation Format: Solomon Levin, Mahsa Shironi, Melissa Jarmel, Michael Tomasini, Bassem Shebl, Tova Finkelstein, David Requena, Soeren Heissel, Henrik Molina, Philip Coffino, Barbara Lyons, Sanford M. Simon. Pathogenesis of fibrolamellar: Proteome and phosphome of an oncokinase driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2494.
Cancer Research, Apr 4, 2023
In fibrolamellar hepatocellular carcinoma (FLC), hyperammonemic encephalopathy is a common occurr... more In fibrolamellar hepatocellular carcinoma (FLC), hyperammonemic encephalopathy is a common occurrence and occasionally causes death. Using mass spectrometry, we quantitatively analyzed the proteomes of FLC patient’s tumor and adjacent normal, to find pathways that are changed in FLC. These data identified multiple proteins that were altered in the proteome, among these, enzymes involved in metabolism of ammonia. These results were confirmed with immunofluorescence demonstrating that these alterations occur in all tumor cells. These results suggest that FLC cells have defects in the two primary ammonia detoxification pathways in the liver, which are responsible for detoxification of 70% of the ammonia in the body: 1) consumption of ammonia by glutamine synthetase (GLUL), and 2) addition of ammonia by ornithine carbamoyltransferase (OTC) to the urea cycle. Additionally, they also generate extra ammonia because of upregulation of glutaminase (GLS). This was tested with a targeted metabolomics of the reactants and products of these enzymes. The results were consistent with both a loss of the two pathways for consumption of ammonia activation of a pathway for generating ammonia. This production of ammonia is consistent with the observation that surgical resection of fibrolamellar reduces the ammonia in patients. All FLC patients with hyperammonemic encephalopathy and documented urine test results showed increased urinary orotic acid, evidence of blockage of the OTC pathway. This study implies that hyperammonemic encephalopathy in FLC may require alternatives to commonly used therapies. Citation Format: Mahsa Shirani, Solomon Levin, Michael D. Tomasini, James Knox, Bassem Shebl, David Requena, Jackson Clark, Søren Heissel, Hanan Alwaseem, Rodrigo Surjan, Ron Lahasky, Henrik Molina, Barbara Lyons, Rachael D. Migler, Philip Coffino, Sanford M. Simon. Urea cycle metabolism is disturbed in Fibrolamellar carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6729.
Supplementary Figure 1 - 6 and Supplementary Tables 1, 2, and 4
To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-d... more To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable.Significance:Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355
Cancer Discovery, Jun 14, 2021
To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-d... more To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A highthroughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein BCL-xL, and inhibition of BCL-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of BCL-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Mar 4, 2011
F1000 - Post-publication peer review of the biomedical literature, 2002
University of California, San Francisco. Archives and Special Collections. Harold E. Varmus Papers, May 26, 1977
To ensure the safety of recombinant DNA research (research that, among many other results, yielde... more To ensure the safety of recombinant DNA research (research that, among many other results, yielded important techniques for Varmus' investigation into the origins of cancer), the National Institutes of Health in 1976 issued guidelines for the construction and operation of laboratories in which such research was conducted. The guidelines established four containment levels, setting more stringent requirement for such features as air exhaust and wastewater disposal systems with each ascending level. This directive lays out the protocol for using and cleaning a level two containment facility in Varmus and Bishop's laboratory.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Apr 2, 2004
Biochemical Journal, Nov 24, 1999
The proteasomes have a central role in catalysing protein degradation among both prokaryotes and ... more The proteasomes have a central role in catalysing protein degradation among both prokaryotes and eukaryotes. The 20 S proteasome constitutes their catalytic core. In studying the structure of Trypanosoma brucei 20 S proteasomes, we isolated by two-dimensional (2D) gel electrophoresis a 27 kDa subunit protein with an estimated pI of 4.7 and subjected it to mass spectrometric analysis. A tryptic peptide sequence from the protein was found identical with that of the rat α5 subunit. With the use of antiserum against T. brucei 20 S proteasomes to screen a T. b. rhodesiense λ expression cDNA library, we obtained a cDNA clone encoding a full-length protein of 246 amino acid residues with a calculated molecular mass of 27 174 Da and a pI of 4.71. It bears 50.0 % and 46.3 % sequence identity with rat and yeast proteasome subunit α5 respectively, and matches all the peptide sequences derived from MS of the 2D gel-purified protein. The protein is thus designated the α5 subunit of T. brucei 20 S proteasome (TbPSA5). The recombinant protein, expressed in plasmid-transformed Escherichia coli, was found in a 27 kDa
European journal of biochemistry, Dec 1, 1991
In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred durin... more In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid-blastula transition (MBT). ODC activity was stable in cycloheximide-treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post-MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC-deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post-translationally by polyamines, indicating that the post-MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine-mediated regulation.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Mar 12, 2013
PubMed, Nov 1, 1976
... SUMMARY INSEL, PAUL A., MAGUIRE, MICHAEL E., GILMAN, ALFRED G., BOURNE, HENRY R., C0FFIN0, PH... more ... SUMMARY INSEL, PAUL A., MAGUIRE, MICHAEL E., GILMAN, ALFRED G., BOURNE, HENRY R., C0FFIN0, PHILIP & MELMON, KENNETH L. (1976) Beta admenergic receptors and adenylate cyclase: products of separate genes? Mol. Pharmacol., 12, 1062-1069. ...
F1000 - Post-publication peer review of the biomedical literature, 2004
Science Advances, Jun 23, 2023
Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a... more Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a somatic dysregulation of protein kinase A. We show that the proteome of FLC tumors is distinct from that of adjacent nontransformed tissue. These changes can account for some of the cell biological and pathological alterations in FLC cells, including their drug sensitivity and glycolysis. Hyperammonemic encephalopathy is a recurrent problem in these patients, and established treatments based on the assumption of liver failure are unsuccessful. We show that many of the enzymes that produce ammonia are increased and those that consume ammonia are decreased. We also demonstrate that the metabolites of these enzymes change as expected. Thus, hyperammonemic encephalopathy in FLC may require alternative therapeutics.
Cancer Research, Jun 15, 2022
Fibrolamellar hepatocellular carcinoma (FLC) is a lethal pediatric cancer affecting adolescents a... more Fibrolamellar hepatocellular carcinoma (FLC) is a lethal pediatric cancer affecting adolescents and young adults. Besides surgical resection, there is no approved and effective therapy. FLC is driven by a fusion oncokinase, DNAJB1-PRKACA, that arises from a deletion fusing exon 1 of DNAJB1 and exons 2-10 of PRKACA, the catalytic subunit of protein kinase A. Expression of this chimeric oncokinase is capable of recapitulating the key histologic and transcriptomic features of FLC. Using PDXs, we tested therapeutics that have been used clinically for FLC or that target pathways identified via the transcriptomic signature of FLC, in addition to an agnostic library of clinical-stage drugs. Several compounds were identified with a varying degree of efficacy in different PDX lines as compared to our control line, primary human hepatocytes. One of the top hits was irinotecan, and its active metabolite SN38. Irinotecan is a topoisomerase I inhibitor and is clinically utilized in a wide variety of adult and pediatric cancers. A second top hit was Navitoclax, an inhibitor of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Tool compounds that targeted Bcl-xL, but not bcl-2 were efficacious. This suggested a possible link between Bcl-xL, which is overexpressed in some PDX lines, and the resistance profile they showed in our screen. Unfortunately, Navitoclax has an on-target and dose-limiting toxicity of thrombocytopenia, which limits its clinical application. DT2216 is a VHL-based PROTAC that degrades Bcl-xL. DT2216 has two potential benefits over Navitoclax: 1) DT2216 is a more potent than Navitoclax against various Bcl-xL dependent tumor cells; and 2) it is less toxic to platelets than Navitoclax by targeting Bcl-xl to VHL that is poorly expressed in platelets to avoid induction of severe thrombocytopenia. A phase 1 clinical trial of DT2216 in relapsed/refractory malignancies is now ongoing (NCT04886622). We tested if the combination of DT2216 and irinotecan might have a therapeutic potential in FLC. Indeed, in vitro screening of the proposed combo, DT2216 and SN38, showed an additive or a synergistical effect in 4 validated FLC cell lines. We tested both the efficacy of DT2216 on Bcl-xL levels in FLC as well as platelet counts to identify any developing thrombocytopenia. DT2216 was less disruptive to platelets than Navitoclax at therapeutically relevant pre-clinical doses. Next, we tested 4 of our PDX models for FLC using various dosing regimens to identify the most efficacious regimen with the minimum side effects. Mice cohorts were evaluated for weight loss and tumor volume. Mice treated with DT2216 and SN38 showed consistent therapeutic benefit which was not evident in controls and less notable as monotherapy. In conclusion, TOPO1 and Bcl-xL prove to be promising targets of interest in FLC. Targeting both with DT2216 and irinotecan leads to tumor shrinkage in pre-clinical models and offer a potential therapeutic/pharmacological intervention for FLC. Citation Format: Bassem Shebl, Gadi Lalazar, Denise Ng, Tova Finkelstein, Guangrong Zheng, Peiyi Zhang, Carly Rosemore, Michael Ortiz, Daohong Zhou, Sanford Simon, Philip Coffino. Targeting Bcl-xL in fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3888.
Cancer Research, Jun 15, 2022
Fibrolamellar hepatocellular carcinoma, FLC, is a usually lethal cancer affecting children, and y... more Fibrolamellar hepatocellular carcinoma, FLC, is a usually lethal cancer affecting children, and young adults. We have shown that all patients have a disruption in the ecology of protein kinase A activity. Hundreds of patients tested have a fusion of the first exon of DNABJ1, a heat shock protein cofactor, to PRKACA, the catalytic subunit of protein kinase A and expression of this DNAJB1-PRKACA fusion oncokinase is sufficient to produce the tumor. One patient is missing the regulatory subunit of protein kinase A and three patients have a fusion of the first exon of a different protein, ATP1B1, to the catalytic subunit of protein kinase A. We characterized the proteome and phosphoproteome of FLC cells relative to adjacent normal tissue. The changes were sufficiently characteristic to identify cell extracts from FLC, based solely on the proteome or phosphoproteome, and distinguishable from other tumor or normal liver. By calibrating protein level we found that tumor cells have an increase of catalytic subunit to such an extent as to exceed the capacity of protein kinase A regulatory subunits. This has two implications. First, there is free catalytic subunit that is unregulated, creating a high basal level of kinase activity. Second, the catalytic subunit is no longer localized by tethering to the regulatory subunit. Thus, the catalytic subunit has access to novel substrates. As an independent confirmation, we found the free basal kinase activity was higher in FLC cells. As a further test, we showed free catalytic subunit, that is not conjugated to regulatory subunit, is higher in FLC cells. We next tested whether these changes were solely due to overexpression of the catalytic subunit or if there was some additional change in the intrinsic activity of the kinase as a result of the fusion. We purified, without using an affinity tag, either PRKACA (which is myristoylated), DNAJB1-PRKACA, and PRKACA which was not myristoylated (the DNAJB1-PRKACA is not myristoylated). As a further control we also used the PRKACAL206R mutation, which does not engage regulatory subunits and is present in some forms of Cushing’s disease. We purified cytosol from human liver, blocked activity of all the endogenous kinases, and added our purified kinase. Mass spectrometry was then used to identify all newly phosphorylated proteins. While many proteins were equally phosphorylated by all four kinases (PRKACA, DNAJB1-PRKACA, PRKACA not myristoylated, PRKACAL206R) there were some distinct differences. From an analysis of these we found alterations in the optimal recognition sequence for phosphorylation for each variant of the kinase. Significantly, when we examined the human tissue data, we found many proteins that were phosphorylated by the DNAJB1-PRKACA in vitro were also phosphorylated in patient tumor tissue, but not in adjacent non-transformed tissue. This validated these observations of altered phosphorylation from the in vitro assays. Citation Format: Solomon Levin, Mahsa Shironi, Melissa Jarmel, Michael Tomasini, Bassem Shebl, Tova Finkelstein, David Requena, Soeren Heissel, Henrik Molina, Philip Coffino, Barbara Lyons, Sanford M. Simon. Pathogenesis of fibrolamellar: Proteome and phosphome of an oncokinase driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2494.
Cancer Research, Apr 4, 2023
In fibrolamellar hepatocellular carcinoma (FLC), hyperammonemic encephalopathy is a common occurr... more In fibrolamellar hepatocellular carcinoma (FLC), hyperammonemic encephalopathy is a common occurrence and occasionally causes death. Using mass spectrometry, we quantitatively analyzed the proteomes of FLC patient’s tumor and adjacent normal, to find pathways that are changed in FLC. These data identified multiple proteins that were altered in the proteome, among these, enzymes involved in metabolism of ammonia. These results were confirmed with immunofluorescence demonstrating that these alterations occur in all tumor cells. These results suggest that FLC cells have defects in the two primary ammonia detoxification pathways in the liver, which are responsible for detoxification of 70% of the ammonia in the body: 1) consumption of ammonia by glutamine synthetase (GLUL), and 2) addition of ammonia by ornithine carbamoyltransferase (OTC) to the urea cycle. Additionally, they also generate extra ammonia because of upregulation of glutaminase (GLS). This was tested with a targeted metabolomics of the reactants and products of these enzymes. The results were consistent with both a loss of the two pathways for consumption of ammonia activation of a pathway for generating ammonia. This production of ammonia is consistent with the observation that surgical resection of fibrolamellar reduces the ammonia in patients. All FLC patients with hyperammonemic encephalopathy and documented urine test results showed increased urinary orotic acid, evidence of blockage of the OTC pathway. This study implies that hyperammonemic encephalopathy in FLC may require alternatives to commonly used therapies. Citation Format: Mahsa Shirani, Solomon Levin, Michael D. Tomasini, James Knox, Bassem Shebl, David Requena, Jackson Clark, Søren Heissel, Hanan Alwaseem, Rodrigo Surjan, Ron Lahasky, Henrik Molina, Barbara Lyons, Rachael D. Migler, Philip Coffino, Sanford M. Simon. Urea cycle metabolism is disturbed in Fibrolamellar carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6729.
Supplementary Figure 1 - 6 and Supplementary Tables 1, 2, and 4
To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-d... more To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable.Significance:Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355
Cancer Discovery, Jun 14, 2021
To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-d... more To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A highthroughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein BCL-xL, and inhibition of BCL-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of BCL-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Mar 4, 2011
F1000 - Post-publication peer review of the biomedical literature, 2002
University of California, San Francisco. Archives and Special Collections. Harold E. Varmus Papers, May 26, 1977
To ensure the safety of recombinant DNA research (research that, among many other results, yielde... more To ensure the safety of recombinant DNA research (research that, among many other results, yielded important techniques for Varmus' investigation into the origins of cancer), the National Institutes of Health in 1976 issued guidelines for the construction and operation of laboratories in which such research was conducted. The guidelines established four containment levels, setting more stringent requirement for such features as air exhaust and wastewater disposal systems with each ascending level. This directive lays out the protocol for using and cleaning a level two containment facility in Varmus and Bishop's laboratory.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Apr 2, 2004
Biochemical Journal, Nov 24, 1999
The proteasomes have a central role in catalysing protein degradation among both prokaryotes and ... more The proteasomes have a central role in catalysing protein degradation among both prokaryotes and eukaryotes. The 20 S proteasome constitutes their catalytic core. In studying the structure of Trypanosoma brucei 20 S proteasomes, we isolated by two-dimensional (2D) gel electrophoresis a 27 kDa subunit protein with an estimated pI of 4.7 and subjected it to mass spectrometric analysis. A tryptic peptide sequence from the protein was found identical with that of the rat α5 subunit. With the use of antiserum against T. brucei 20 S proteasomes to screen a T. b. rhodesiense λ expression cDNA library, we obtained a cDNA clone encoding a full-length protein of 246 amino acid residues with a calculated molecular mass of 27 174 Da and a pI of 4.71. It bears 50.0 % and 46.3 % sequence identity with rat and yeast proteasome subunit α5 respectively, and matches all the peptide sequences derived from MS of the 2D gel-purified protein. The protein is thus designated the α5 subunit of T. brucei 20 S proteasome (TbPSA5). The recombinant protein, expressed in plasmid-transformed Escherichia coli, was found in a 27 kDa
European journal of biochemistry, Dec 1, 1991
In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred durin... more In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid-blastula transition (MBT). ODC activity was stable in cycloheximide-treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post-MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC-deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post-translationally by polyamines, indicating that the post-MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine-mediated regulation.