Microdissected Pyramidal Cell Proteomics of Alzheimer Brain Reveals Alterations in Creatine Kinase B-Type, 14-3-3-γ, and Heat Shock Cognate 71

Novel insights on proteins involved in Alzheimer’s disease (AD) are needed. Since multiple cell types and matrix components are altered in AD, bulk analysis of brain tissue maybe difficult to interpret. In the current study, we isolated pyramidal cells from the cornu ammonis 1 (CA1) region of the hippocampus from five AD and five neurologically healthy donors using laser capture microdissection (LCM). The samples were analyzed by proteomics using 18O-labeled internal standard and nano-high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for relative quantification. Fold change between AD and control was calculated for the proteins that were identified in at least two individual proteomes from each group. From the 10 cases analyzed, 62 proteins were identified in at least two AD cases and two control cases. Creatine kinase B-type (CKB), 14-3-3-γ, and heat shock cognate 71 (Hsc71), which have not been extensively studied in the context of the human AD brain previously, were selected for further studies by immunohistochemistry (IHC). In hippocampus, semi-quantitative measures of IHC staining of the three proteins confirmed the findings from our proteomic analysis. Studies of the same proteins in the frontal cortex revealed that the alterations remained for CKB and 14-3-3-γ but not for Hsc71. Protein upregulation in CA1 neurons of final stage AD is either a result of detrimental, pathological effects, or from cell-specific protective response mechanisms in surviving neurons. Based on previous findings from experimental studies, CKB and Hsc71 likely exhibit protective effects, whereas 14-3-3-γ may represent a detrimental pathway. These new players could reflect pathways of importance for the development of new therapeutic strategies.

Therapeutic targeting of SLC6A8 creatine transporter inhibits KRAS mutant and wildtype colon cancer and modulates human creatine levels

ABSTRACTColorectal cancer (CRC) is a leading cause of cancer mortality. Creatine metabolism was previously shown to critically regulate colon cancer progression. We report that RGX-202, an oral small-molecule SLC6A8 creatine transporter inhibitor, robustly inhibits creatine import in vitro and in vivo, reduces intracellular phosphocreatine and ATP levels and induces tumor cell apoptosis in CRC. RGX-202 suppressed tumor growth across KRAS wild-type and KRAS mutant xenograft, syngeneic and patient-derived xenograft colorectal cancers. Anti-tumor efficacy correlated with tumoral expression of creatine kinase B. Combining RGX-202 with 5- fluorouracil or the DHODH inhibitor leflunomide caused regressions of multiple colorectal xenograft and PDX tumors of distinct mutational backgrounds. RGX-202 also perturbed creatine metabolism in metastatic CRC patients enrolled in a Phase-1 trial, mirroring pharmacodynamic effects on creatine metabolism observed in mice. This is, to our knowledge, the first demonstration of pre-clinical and human pharmacodynamic activity for creatine metabolism targeting in oncology, revealing a critical target for CRC.

Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B

Inositol hexakisphosphate kinases (IP6Ks) regulate various biological processes. IP6Ks convert IP6 to pyrophosphates such as diphosphoinositol pentakisphosphate (IP7) and bis-diphosphoinositol tetrakisphosphate (IP8). IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, IP6K1, IP6K2, and IP6K3, which have distinct biological functions. The inositol hexakisphosphate kinase 2 (IP6K2) controls cellular apoptosis. To explore roles for IP6K2 in brain function, we elucidated its protein interactome in mouse brain revealing a robust association of IP6K2 with creatine kinase-B (CK-B), a key enzyme in energy homeostasis. Cerebella of IP6K2-deleted mice (IP6K2-knockout [KO]) produced less phosphocreatine and ATP and generated higher levels of reactive oxygen species and protein oxidative damage. In IP6K2-KO mice, mitochondrial dysfunction was associated with impaired expression of the cytochrome-c1 subunit of complex III of the electron transport chain. We reversed some of these effects by combined treatment with N-acetylcysteine and phosphocreatine. These findings establish a role for IP6K2–CK-B interaction in energy homeostasis associated with neuroprotection.

Phase I monotherapy dose escalation of RGX-202, a first-in-class oral inhibitor of the SLC6a8/CKB pathway, in patients with advanced gastrointestinal (GI) solid tumors.

3504 Background: About 65% of advanced colorectal cancer (CRC) patients (pts) have creatine kinase B (CKB) expressing tumors. CKB expressing (CKB+) GI cancer cells import creatine via the creatine transporter SLC6a8 and utilize it to generate intracellular ATP. RGX-202, a small molecule inhibitor of SLC6a8, reduces intracellular creatine and ATP levels, leading to apoptosis. RGX-202 treatment triggers complete tumor regressions in multiple CKB+ preclinical models, including KRAS mutant CRC. Methods: RGX-202-001 is a phase I escalation/expansion study of RGX-202 +/- FOLFIRI in pts with advanced GI tumors. The primary safety objective during dose escalation is to identify the maximum tolerated dose (MTD), or the maximum tested dose at which multiple dose-limiting toxicities (DLTs) are not observed. The primary efficacy objective is to estimate the antitumor activity of RGX-202 by RECIST. Results: As of January 31, 2020, 17 pts have been treated in 4 single agent dose escalation cohorts: 600 mg BID (3 pts), 1200 mg BID (4 pts), 2400mg BID (5 pts) and 3600mg BID (5pts) given continuously. No DLTs were observed and an MTD was not reached. Treatment-related adverse events (TRAEs) occurring in > 2 pts are shown in the Table. There were no Grade 4 TRAEs. At the highest dose, 2 of 3 CRC pts had prolonged disease control: a patient with a KRAS G13D mutant cancer had SD for 14 weeks; and a patient with KRAS G12V mutant (MSS) cancer had a confirmed PR ongoing at 30 weeks. Exposure to RGX-202 was greater than dose-proportional and the average AUC0-24 ranged from ~15,700 ng-hr/mL in cohort 1 to 241,097 ng-hr/mL in in Cohort 4. Serum and urine creatine levels, pharmacodynamic markers of SLC6a8 inhibition, correlated with systemic exposure to RGX-202. Conclusions: Among 17 patients treated with single agent therapy, no DLTs occurred; notably, exposures predicted to be sufficient to inhibit human tumor growth from preclinical models were achieved along with concomitant pharmacodynamic effects. These data, along with a durable PR observed in the highest dose cohort, support further development of RGX-202. Consequently, dose escalation in combination with FOLFIRI in patients with advanced GI cancers is underway with plans for expansion in CKB+ CRC pts. Clinical trial information: NCT03597581 . [Table: see text]