Durvalumab With or Without Tremelimumab for Patients With Metastatic Pancreatic Ductal Adenocarcinoma: A Phase 2 Randomized Clinical Trial.

IMPORTANCE: New therapeutic options for patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) are needed. This study evaluated dual checkpoint combination therapy in patients with mPDAC. OBJECTIVE: To evaluate the safety and efficacy of the anti-PD-L1 (programmed death-ligand 1) antibody using either durvalumab monotherapy or in combination with the anticytotoxic T-lymphocyte antigen 4 antibody using durvalumab plus tremelimumab therapy in patients with mPDAC. DESIGN, SETTING, AND PARTICIPANTS: Part A of this multicenter, 2-part, phase 2 randomized clinical trial was a lead-in safety, open-label study with planned expansion to part B pending an efficacy signal from part A. Between November 26, 2015, and March 23, 2017, 65 patients with mPDAC who had previously received only 1 first-line fluorouracil-based or gemcitabine-based treatment were enrolled at 21 sites in 6 countries. Efficacy analysis included the intent-to-treat population; safety analysis included patients who received at least 1 dose of study treatment and for whom any postdose data were available. INTERVENTIONS: Patients received durvalumab (1500 mg every 4 weeks) plus tremelimumab (75 mg every 4 weeks) combination therapy for 4 cycles followed by durvalumab therapy (1500 mg every 4 weeks) or durvalumab monotherapy (1500 mg every 4 weeks) for up to 12 months or until the onset of progressive disease or unacceptable toxic effects. MAIN OUTCOMES AND MEASURES: Safety and efficacy were measured by objective response rate, which was used to determine study expansion to part B. The threshold for expansion was an objective response rate of 10% for either treatment arm. RESULTS: Among 65 randomized patients, 34 (52%) were men and median age was 61 (95% CI, 37-81) years. Grade 3 or higher treatment-related adverse events occurred in 7 of 32 patients (22%) receiving combination therapy and in 2 of 32 patients (6%) receiving monotherapy; 1 patient randomized to the monotherapy arm did not receive treatment owing to worsened disease. Fatigue, diarrhea, and pruritus were the most common adverse events in both arms. Overall, 4 of 64 patients (6%) discontinued treatment owing to treatment-related adverse events. Objective response rate was 3.1% (95% CI, 0.08-16.22) for patients receiving combination therapy and 0% (95% CI, 0.00-10.58) for patients receiving monotherapy. Low patient numbers limited observation of the associations between treatment response and PD-L1 expression or microsatellite instability status. CONCLUSION AND RELEVANCE: Treatment was well tolerated, and the efficacy of durvalumab plus tremelimumab therapy and durvalumab monotherapy reflected a population of patients with mPDAC who had poor prognoses and rapidly progressing disease. Patients were not enrolled in part B because the threshold for efficacy was not met in part A. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02558894.

Ramucirumab as Second-Line Treatment in Patients With Advanced Hepatocellular Carcinoma: Analysis of REACH Trial Results by Child-Pugh Score.

IMPORTANCE: REACH is the first phase 3 trial to provide information on hepatocellular cancer (HCC) in the second-line (postsorafenib) setting categorized by Child-Pugh score, a scoring system used to measure the severity of chronic liver disease. This exploratory analysis demonstrates the relationship between a potential ramucirumab survival benefit, severity of liver disease, and baseline α-fetoprotein (αFP). OBJECTIVE: To assess treatment effects and tolerability of ramucirumab by Child-Pugh score in patients with HCC enrolled in the REACH trial. DESIGN, SETTINGS, AND PARTICIPANTS: Randomized, double-blind, phase 3 trial of ramucirumab and best supportive care vs placebo and best supportive care as second-line treatment in patients with HCC enrolled between November 4, 2010 and April 18, 2013, from 154 global sites. Overall, 643 patients were randomized and included in this analysis; 565 patients considered Child-Pugh class A (Child-Pugh scores 5 and 6) and 78 patients considered class B (Child-Pugh scores 7 and 8). INTERVENTIONS: Ramucirumab (8 mg/kg) or placebo intravenously plus best supportive care every 2 weeks. MAIN OUTCOMES AND MEASURES: Overall survival (OS), defined as time from randomization to death from any cause. RESULTS: In the randomized population of 643 patients (mean [SD] age, 62.8 [11.1] years) in this analysis, a potential ramucirumab OS benefit was observed for patients with a Child-Pugh score of 5 (hazard ratio [HR], 0.80; 95% CI, 0.63-1.02; P = .06) but no apparent benefit for patients with Child-Pugh scores of 6 or 7 and 8. In patients with baseline αFP levels of 400 ng/mL (to convert ng/mL to µg/L, multiply by 1.0) or more, a ramucirumab OS benefit was significant for a score of Child-Pugh 5 (HR, 0.61; 95% CI, 0.43-0.87; P = .01) and Child-Pugh 6 (HR, 0.64; 95% CI, 0.42-0.98; P = .04), but was not significant for Child-Pugh 7 and 8. The overall safety profile of ramucirumab, regardless of Child-Pugh score, was considered manageable. Regardless of treatment arm, patients with Child-Pugh scores of 7 and 8 experienced a higher incidence of grade 3 or higher treatment-emergent adverse events, including ascites and asthenia, and special-interest events, including liver injury and/or failure and bleeding, compared with patients with Child-Pugh scores of 5 or 6. CONCLUSIONS AND RELEVANCE: In unselected patients, a trend for ramucirumab survival benefit was observed only for patients with a Child-Pugh score of 5. In patients with baseline αFP levels of 400 ng/mL or more, a ramucirumab survival benefit was observed for Child-Pugh scores of 5 and 6. Ramucirumab had a manageable toxic effect profile. These results support the ongoing REACH-2 study of ramucirumab in patients with advanced HCC with underlying Child-Pugh A cirrhosis and baseline αFP levels of 400 ng/mL or more. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01140347.

Cetuximab and Radiotherapy in Laryngeal Preservation for Cancers of the Larynx and Hypopharynx: A Secondary Analysis of a Randomized Clinical Trial.

IMPORTANCE: The appropriate use of surgery or radiotherapy-based approaches for organ preservation has been the subject of much debate. Unfortunately, there has been a lack of improvement in overall survival for patients with laryngeal carcinoma in the last 30 years. OBJECTIVE: To assess the rates of laryngeal preservation and laryngectomy-free survival in patients receiving cetuximab and radiotherapy (CRT) and patients receiving radiotherapy alone. DESIGN, SETTING, AND PARTICIPANTS: Patients were enrolled in a multicenter, open-label, stratified, randomized, phase 3 study from April 1, 1999, through March 31, 2002, from 73 centers in the United States and 14 other countries. A secondary subgroup analysis of patients with hypopharyngeal and laryngeal carcinoma was undertaken. Rates of laryngeal preservation and laryngectomy-free survival were estimated by the Kaplan-Meier method. The hazard ratios (HRs) were calculated using a Cox proportional hazards regression model. Quality of life was evaluated using the European Organization for Research and Treatment of Cancer core questionnaire and head and neck module. MAIN OUTCOMES AND MEASURES: Laryngeal preservation and laryngectomy-free survival. RESULTS: Of the 424 patients included in the trial, 168 treated patients with cancer of the larynx or hypopharynx were included in this analysis (90 in the CRT group and 78 in the radiotherapy alone group). The median (range) age of the patients was 59 (40-80) years in the CRT group and 61 (35-81) years in the radiotherapy alone group. In the CRT group, 72 patients (80.0%) were male and 18 (20.0%) were female. In the radiotherapy alone group, 62 (79.5%) were male and 16 (20.5%) were female. The rates of laryngeal preservation at 2 years were 87.9% for CRT vs 85.7% for radiotherapy alone, with an HR of 0.57 (95% CI, 0.23-1.42; P = .22). Similarly, the HR for laryngectomy-free survival comparing CRT vs radiotherapy alone was 0.78 (95% CI, 0.54-1.11; P = .17). This study was not powered to assess organ preservation. Median overall survival was 27 (95% CI, 20-45) vs 21 (95% CI, 17-35) months for the CRT and radiotherapy alone groups, respectively, with an HR of 0.87 (95% CI, 0.60-1.27). No differences between treatments were reported regarding overall quality of life, need for a feeding tube, or speech. CONCLUSIONS AND RELEVANCE: The results of a possible cetuximab-related laryngeal preservation benefit for patients with hypopharyngeal or laryngeal cancer are intriguing; these results need to be interpreted in the context of a retrospective subset analysis with limited sample size. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00004227.

Cetuximab plus platinum-based chemotherapy in head and neck squamous cell carcinoma: a randomized, double-blind safety study comparing cetuximab produced from two manufacturing processes using the EXTREME study regimen.

BACKGROUND: Cetuximab, in combination with platinum chemotherapy plus 5-fluoruracil (5-FU), is approved for the first-line treatment of recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN). Cetuximab manufactured by ImClone (US commercial cetuximab) potentially results in higher systemic exposures than cetuximab manufactured by Boehringer Ingelheim (BI-manufactured cetuximab). This prospective, randomized, double-blind study compared the safety profiles of the two cetuximab formulations. METHODS: Patients with previously untreated locoregionally recurrent and/or metastatic SCCHN were randomly assigned to receive the same dose of US commercial cetuximab (Arm A) or BI-manufactured cetuximab (Arm B), each in combination with cisplatin or carboplatin plus 5-FU. The primary outcome was all-grade, all-cause treatment-emergent adverse events (TEAEs). RESULTS: The majority of patients experienced ≥ 1 TEAE, regardless of causality (Arm A: 75/77 patients, 97.4%; Arm B: 68/71 patients, 95.8%). TEAEs with the highest incidence included nausea, fatigue, and hypomagnesemia in both arms. The absolute risk difference between the two arms for patients experiencing at least one adverse event (AE) was 0.029 (p = 0.281, 95% confidence interval [CI]: -0.024, 0.082) for AEs regardless of causality and 0.005 (p = 0.915, 95% CI: -0.092, 0.103) for AEs possibly related to study drug. There were no significant differences between the two arms in the incidence of acneiform rash, cardiac events, infusion reactions, or hypomagnesemia. Overall survival, progression-free survival, and overall response rates were similar in the two arms. CONCLUSIONS: There were no clinically meaningful differences in safety between US commercial cetuximab and BI-manufactured cetuximab in combination with platinum-based therapy with 5-FU in patients with locoregionally recurrent and/or metastatic SCCHN. The use of US commercial cetuximab in this combination chemotherapy regimen did not result in any unexpected safety signals. The efficacy results of this study are consistent with the efficacy results of the cetuximab arm of the EXTREME study. TRIAL REGISTRATION: ClinicalTrials.gov NCT01081041 ; date of registration: March 3, 2010).

Ramucirumab versus placebo as second-line treatment in patients with advanced hepatocellular carcinoma following first-line therapy with sorafenib (REACH): a randomised, double-blind, multicentre, phase 3 trial.

BACKGROUND: VEGF and VEGF receptor-2-mediated angiogenesis contribute to hepatocellular carcinoma pathogenesis. Ramucirumab is a recombinant IgG1 monoclonal antibody and VEGF receptor-2 antagonist. We aimed to assess the safety and efficacy of ramucirumab in advanced hepatocellular carcinoma following first-line therapy with sorafenib. METHODS: In this randomised, placebo-controlled, double-blind, multicentre, phase 3 trial (REACH), patients were enrolled from 154 centres in 27 countries. Eligible patients were aged 18 years or older, had hepatocellular carcinoma with Barcelona Clinic Liver Cancer stage C disease or stage B disease that was refractory or not amenable to locoregional therapy, had Child-Pugh A liver disease, an Eastern Cooperative Oncology Group performance status of 0 or 1, had previously received sorafenib (stopped because of progression or intolerance), and had adequate haematological and biochemical parameters. Patients were randomly assigned (1:1) to receive intravenous ramucirumab (8 mg/kg) or placebo every 2 weeks, plus best supportive care, until disease progression, unacceptable toxicity, or death. Randomisation was stratified by geographic region and cause of liver disease with a stratified permuted block method. Patients, medical staff, investigators, and the funder were masked to treatment assignment. The primary endpoint was overall survival in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01140347. FINDINGS: Between Nov 4, 2010, and April 18, 2013, 565 patients were enrolled, of whom 283 were assigned to ramucirumab and 282 were assigned to placebo. Median overall survival for the ramucirumab group was 9·2 months (95% CI 8·0-10·6) versus 7·6 months (6·0-9·3) for the placebo group (HR 0·87 [95% CI 0·72-1·05]; p=0·14). Grade 3 or greater adverse events occurring in 5% or more of patients in either treatment group were ascites (13 [5%] of 277 patients treated with ramucirumab vs 11 [4%] of 276 patients treated with placebo), hypertension (34 [12%] vs ten [4%]), asthenia (14 [5%] vs five [2%]), malignant neoplasm progression (18 [6%] vs 11 [4%]), increased aspartate aminotransferase concentration (15 [5%] vs 23 [8%]), thrombocytopenia (13 [5%] vs one [<1%]), hyperbilirubinaemia (three [1%] vs 13 [5%]), and increased blood bilirubin (five [2%] vs 14 [5%]). The most frequently reported (≥1%) treatment-emergent serious adverse event of any grade or grade 3 or more was malignant neoplasm progression. INTERPRETATION: Second-line treatment with ramucirumab did not significantly improve survival over placebo in patients with advanced hepatocellular carcinoma. No new safety signals were noted in eligible patients and the safety profile is manageable. FUNDING: Eli Lilly and Co.

Ramucirumab versus placebo in combination with second-line FOLFIRI in patients with metastatic colorectal carcinoma that progressed during or after first-line therapy with bevacizumab, oxaliplatin, and a fluoropyrimidine (RAISE): a randomised, double-blind, multicentre, phase 3 study.

BACKGROUND: Angiogenesis is an important therapeutic target in colorectal carcinoma. Ramucirumab is a human IgG-1 monoclonal antibody that targets the extracellular domain of VEGF receptor 2. We assessed the efficacy and safety of ramucirumab versus placebo in combination with second-line FOLFIRI (leucovorin, fluorouracil, and irinotecan) for metastatic colorectal cancer in patients with disease progression during or after first-line therapy with bevacizumab, oxaliplatin, and a fluoropyrimidine. METHODS: Between Dec 14, 2010, and Aug 23, 2013, we enrolled patients into the multicentre, randomised, double-blind, phase 3 RAISE trial. Eligible patients had disease progression during or within 6 months of the last dose of first-line therapy. Patients were randomised (1:1) via a centralised, interactive voice-response system to receive 8 mg/kg intravenous ramucirumab plus FOLFIRI or matching placebo plus FOLFIRI every 2 weeks until disease progression, unacceptable toxic effects, or death. Randomisation was stratified by region, KRAS mutation status, and time to disease progression after starting first-line treatment. The primary endpoint was overall survival in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01183780.ld FINDINGS: We enrolled 1072 patients (536 in each group). Median overall survival was 13·3 months (95% CI 12·4-14·5) for patients in the ramucirumab group versus 11·7 months (10·8-12·7) for the placebo group (hazard ratio 0·844 95% CI 0·730-0·976; log-rank p=0·0219). Survival benefit was consistent across subgroups of patients who received ramucirumab plus FOLFIRI. Grade 3 or worse adverse events seen in more than 5% of patients were neutropenia (203 [38%] of 529 patients in the ramucirumab group vs 123 [23%] of 528 in the placebo group, with febrile neutropenia incidence of 18 [3%] vs 13 [2%]), hypertension (59 [11%] vs 15 [3%]), diarrhoea (57 [11%] vs 51 [10%]), and fatigue (61 [12%] vs 41 [8%]). INTERPRETATION: Ramucirumab plus FOLFIRI significantly improved overall survival compared with placebo plus FOLFIRI as second-line treatment for patients with metastatic colorectal carcinoma. No unexpected adverse events were identified and toxic effects were manageable. FUNDING: Eli Lilly.

Comparison of KRAS genotype: therascreen assay vs. LNA-mediated qPCR clamping assay.

BACKGROUND: Kirsten rat sarcoma virus (KRAS) wild-type status determined using a locked nucleic acid (LNA)-mediated quantitative polymerase chain reaction (qPCR) clamping assay (LNA assay) predicted response to therapy in the CRYSTAL (Cetuximab Combined With Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer) study. A companion KRAS diagnostic tool has been developed for routine clinical use (QIAGEN therascreen kit) (QIAGEN Manchester Ltd, Manchester, UK). We wanted to assess the concordance between the validated US Food and Drug Administration (FDA)-approved therascreen assay and the LNA assay in determining the KRAS status of a subset of patients enrolled in the CRYSTAL study. PATIENTS AND METHODS: DNA extracted from paraffin-embedded tumor sections was tested for KRAS status using the therascreen assay. Efficacy data from the CRYSTAL study were assessed to determine if the overall survival (OS) hazard ratio for cetuximab in patients identified as having KRAS wild-type status using the therascreen assay was equivalent to that in patients identified as KRAS wild-type using the LNA assay. This was determined by assessing if the concordance between the therascreen assay and the LNA assay met the minimum threshold (prespecified as 0.8) to achieve a significant difference in the OS hazard ratio in favor of the cetuximab + FOLFIRI (5-fluorouracil, leucovorin [folinic acid], irinotecan) arm in the KRAS wild-type population as identified using the therascreen assay. RESULTS: Of the 148 samples determined to be KRAS wild-type (therascreen assay), 141 (95.3%) samples were also KRAS wild-type (LNA assay) and 7 samples (4.7%) were KRAS mutant (LNA assay). The prespecified primary concordance measure p was 141/148 = 0.953 (95% confidence interval [CI], 0.905-0.981). The concordance was statistically significantly higher than the prespecified threshold of 0.8 for concordance between the therascreen assay and the LNA assay. Consistent with the concordance exceeding the prespecified threshold, the OS hazard ratio (cetuximab + FOLFIRI arm vs. FOLFIRI arm) in the KRAS wild-type population, determined by the therascreen assay, supported a significant benefit for cetuximab (ie, the 95% CI excluded 1) and was comparable to the OS hazard ratio observed in the CRYSTAL study KRAS wild-type population (LNA assay) even after adjustment for potentially confounding baseline variables. CONCLUSION: These results support the utility of the therascreen assay for identifying patients who may benefit from cetuximab therapy for metastatic colorectal cancer.

Validation of companion diagnostic for detection of mutations in codons 12 and 13 of the KRAS gene in patients with metastatic colorectal cancer: analysis of the NCIC CTG CO.17 trial.

CONTEXT: The therascreen KRAS RGQ polymerase chain reaction kit is being developed as a companion diagnostic to aid clinicians, through detection of KRAS mutations, in the identification of patients with metastatic colorectal cancer (mCRC) who are more likely to benefit from cetuximab. OBJECTIVES: To assess whether KRAS mutation status, determined by using the therascreen KRAS kit, is a predictive marker of cetuximab efficacy. DESIGN: Tissue samples were obtained from patients with mCRC treated on the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) CO.17 phase 3 study of cetuximab plus best supportive care (BSC) versus BSC alone. Tumor DNA samples were assessed for the presence of KRAS mutations by using the therascreen KRAS kit. Efficacy and safety were assessed to determine whether mutation status was predictive of outcomes. Results.-Evaluable samples were available from 453 patients (79.2%) enrolled in the NCIC CTG CO.17 trial. The KRAS wild-type subset represented 54.1% (245 of 453) of the evaluated population. Median overall survival of patients with KRAS wild-type tumors was 8.6 months among those who received cetuximab plus BSC and 5.0 months among patients who received BSC alone (hazard ratio [HR], 0.63; P = .002). Among patients with KRAS mutant mCRC, no meaningful difference in overall survival was observed between arms (HR, 0.91; P = .55). These results are consistent with a previous report that analyzed patient tumor samples by using bidirectional sequencing. CONCLUSIONS: These data support the utility of the therascreen KRAS kit as a means of selecting patients who may benefit from cetuximab therapy.

Pemetrexed in combination with cisplatin versus cisplatin monotherapy in patients with recurrent or metastatic head and neck cancer: final results of a randomized, double-blind, placebo-controlled, phase 3 study.

BACKGROUND: Recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) is associated with poor survival. Platinum-based chemotherapy is often a first-line treatment. Pemetrexed has shown single-agent activity in SCCHN and in combination with cisplatin for other tumors. This trial examined the efficacy of pemetrexed-cisplatin for SCCHN. METHODS: In a double-blind phase 3 trial, patients with recurrent or metastatic SCCHN and no prior systemic therapy for metastatic disease were randomized to pemetrexed (500 mg/m(2) ) plus cisplatin (75 mg/m(2) ; n = 398) or placebo plus cisplatin (75 mg/m(2) ; n = 397) to assess overall survival (OS) and secondary endpoints. RESULTS: Median OS was 7.3 months in the pemetrexed-cisplatin arm and 6.3 months in the placebo-cisplatin arm (hazard ratio [HR], 0.87; 95% confidence interval [CI], 0.75-1.02; P = .082). Median progression-free survival (PFS, months) was similar in both treatment arms (pemetrexed-cisplatin, 3.6; placebo-cisplatin, 2.8; HR, 0.88; 95% CI, 0.76-1.03; P = .166). Among patients with performance status 0 or 1, pemetrexed-cisplatin (n = 347) led to longer OS and PFS than placebo-cisplatin (n = 343; 8.4 vs 6.7 months; HR, 0.83; P = .026; 4.0 vs 3.0 months; HR, 0.84; P = .044, respectively). Among patients with oropharyngeal cancers, pemetrexed-cisplatin (n = 86) resulted in longer OS and PFS than placebo-cisplatin (n = 106; 9.9 vs 6.1 months; HR, 0.59; P = .002; 4.0 vs 3.4 months; HR, 0.73; P = .047, respectively). Pemetrexed-cisplatin toxicity was consistent with studies in other tumors. CONCLUSIONS: Pemetrexed-cisplatin compared with placebo-cisplatin did not significantly improve survival for the intent-to-treat population. However, in a prespecified subgroup analysis, pemetrexed-cisplatin showed OS and PFS advantage for patients with performance status 0 or 1 or oropharyngeal cancers.

MTMR9 increases MTMR6 enzyme activity, stability, and role in apoptosis.

Myotubularin-related protein 6 (MTMR6) is a catalytically active member of the myotubularin (MTM) family, which is composed of 14 proteins. Catalytically active myotubularins possess 3-phosphatase activity dephosphorylating phosphatidylinositol-3-phoshate and phosphatidylinositol-3,5-bisphosphate, and some members have been shown to form homomers or heteromeric complexes with catalytically inactive myotubularins. We demonstrate that human MTMR6 forms a heteromer with an enzymatically inactive member myotubularin-related protein 9 (MTMR9), both in vitro and in cells. MTMR9 increased the binding of MTMR6 to phospholipids without changing the lipid binding profile. MTMR9 increased the 3-phosphatase activity of MTMR6 up to 6-fold. We determined that MTMR6 is activated up to 28-fold in the presence of phosphatidylserine liposomes. Together, MTMR6 activity in the presence of MTMR9 and assayed in phosphatidylserine liposomes increased 84-fold. Moreover, the formation of this heteromer in cells resulted in increased protein levels of both MTMR6 and MTMR9, probably due to the inhibition of degradation of both proteins. Furthermore, co-expression of MTMR6 and MTMR9 decreased etoposide-induced apoptosis, whereas decreasing both MTMR6 and MTMR9 by RNA interference led to increased cell death in response to etoposide treatment when compared with that seen with RNA interference of MTMR6 alone. Thus, MTMR9 greatly enhances the functions of MTMR6.

Inositol polyphosphate multikinase regulates inositol 1,4,5,6-tetrakisphosphate.

The human inositol phosphate multikinase (IPMK, 5-kinase) has a preferred 5-kinase activity over 3-kinase and 6-kinase activities and a substrate preference for inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) over inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). We now report that the recombinant human protein can catalyze the conversion of inositol 1,4,5,6-tetrakisphosphate (Ins(1,4,5,6)P4) to Ins(1,3,4,5,6)P5 in vitro; the reaction product was identified by HPLC to be Ins(1,3,4,5,6)P5. The apparent Vmax was 42 nmol of Ins(1,3,4,5,6)P5 formed/min/mg protein, and the apparent Km was 222 nM using Ins(1,3,4,6)P4 as a substrate; the catalytic efficiency was similar to that for Ins(1,4,5)P3. Stable over-expression of the human protein in HEK-293 cells abrogates the in vivo elevation of Ins(1,4,5,6)P4 from the Salmonella dublin SopB protein. Hence, the human 5-kinase may also regulate the level of Ins(1,4,5,6)P4 and have an effect on chloride channel regulation.

The identification and characterization of two phosphatidylinositol-4,5-bisphosphate 4-phosphatases.

Numerous inositol polyphosphate 5-phosphatases catalyze the degradation of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P(2)) to phosphatidylinositol-4-phosphate (PtdIns-4-P). An alternative pathway to degrade PtdIns-4,5-P(2) is the hydrolysis of PtdIns-4,5-P(2) by a 4-phosphatase, leading to the production of PtdIns-5-P. Whereas the bacterial IpgD enzyme is known to catalyze this reaction, no such mammalian enzyme has been found. We have identified and characterized two previously undescribed human enzymes, PtdIns-4,5-P(2) 4-phosphatase type I and type II, which catalyze the hydrolysis of PtdIns-4,5-P(2) to phosphatidylinositol-5-phosphate (PtdIns-5-P). Both enzymes are ubiquitously expressed and localize to late endosomal/lysosomal membranes in epithelial cells. Overexpression of either enzyme in HeLa cells increases EGF-receptor degradation upon EGF stimulation.

The pathway for the production of inositol hexakisphosphate in human cells.

The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

The human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase.

We have demonstrated that the human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase (InsP(4) 5-kinase). The cDNA of the human gene contained a putative open reading frame of 1251 bp encoding 416 amino acids with 83.6% identity compared with the rat protein. The substrate specificity of the recombinant human protein demonstrated preference for Ins(1,3,4,6)P(4) with a catalytic efficiency (V(max)/K(m)) 43-fold greater than that of Ins(1,3,4,5)P(4) and 2-fold greater than that of Ins(1,4,5)P(3). The apparent V(max) was 114 nmol of Ins(1,3,4,5,6)P(5) formed/min/mg of protein, and the apparent K(m) was 0.3 microm Ins(1,3,4,6)P(4). The functional homolog in yeast is Ipk2p, and ipk2-null yeast strains do not synthesize Ins(1,3,4,5,6)P(5) or InsP(6). Synthesis of these compounds was restored by transformation with wild-type yeast IPK2 but not with human InsP(4) 5-kinase. Thus the human gene does not complement for the loss of the yeast gene because yeast cells do not contain the substrate Ins(1,3,4,6)P(4), and the reaction of the human protein with Ins(1,3,4,5)P(4) is insufficient to effect rescue or synthesis of InsP(5) and InsP(6). Therefore the major activity of human InsP(4) 5-kinase is phosphorylation at the D-5 position, and the pathways for synthesis of Ins(1,3,4,5,6)P(5) in yeast versus humans are different.