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Preclinical models suggest anticancer activity of IM156, a novel biguanide mitochondrial protein complex 1 inhibitor of oxidative phosphorylation (OXPHOS). This first-in-human dose-escalation study enrolled patients with refractory advanced solid tumors to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). Eligible patients received oral IM156 every other day (QOD) or daily (QD) and were assessed for safety, dose-limiting toxicities (DLTs), pharmacokinetics, and preliminary signals of efficacy. 22 patients with advanced cancers (gastric, n = 8; colorectal, n = 3; ovarian, n = 3; other, n = 8) received IM156 100 to 1,200 mg either QOD or QD. There were no DLTs. However, 1,200 mg QD was not well tolerated due to nausea; 800 mg QD was determined as the RP2D. The most frequent treatment-related AEs (TRAEs) were nausea (n = 15; 68%), diarrhea (n = 10; 46%), emesis (n = 9; 41%), fatigue (n = 4; 18%) and abdominal pain, constipation, and blood lactate increased (n = 2 each; 9%). Grade 3 nausea (n = 3; 14%) was the only grade ≥ 3 TRAE. Plasma exposures increased dose proportionally; mean Day 27 area under the curve (AUC0-24) values were higher following QD administration compared to the respective QOD regimen. Stable disease (SD), observed in 7 (32%) patients (confirmed in 2 [9%]), was the best response. To our knowledge, this is the first phase 1 study of an OXPHOS inhibitor that established a RP2D for further clinical development in cancer. Observed AEs of IM156 were manageable and SD was the best response.

Metabolic reprogramming of the myofibroblast plays a fundamental role in the pathogenesis of fibrosing interstitial lung diseases. Here, we characterized the in vitro and in vivo metabolic and anti-fibrotic effects of IM156, an oxidative phosphorylation (OXPHOS) modulator that acts by inhibiting Protein Complex 1 (PC1). In vitro, IM156 inhibited TGFβ-dependent increases in mitochondrial oxygen consumption rate and expression of myofibroblast markers in human pulmonary fibroblasts without altering cell viability or adding to TGF-β induced increases in the extracellular acidification rate (ECAR). IM156 significantly increased cellular AMPK phosphorylation and was 60-fold more potent than metformin. In vivo, chronic oral administration of IM156 was highly distributed to major peripheral organs (i.e. lung, liver, kidney, heart) and had significant dose-related effects on the plasma metabolome consistent with OXPHOS modulation and AMPK activation. IM156 increased glycolysis, lipolysis, β-oxidation and amino acids, and decreased free fatty acids, TCA cycle activity and protein synthesis. In the murine bleomycin model of pulmonary fibrosis, daily oral administration of IM156 administered 7 days after lung injury, attenuated body/lung weight changes, and reduced lung fibrosis and inflammatory cell infiltration. The plasma exposures of IM156 were comparable to well-tolerated doses in human studies. In conclusion, the metabolic and anti-fibrotic effects of IM156 suggest that OXPHOS modulation can attenuate myofibroblast metabolic reprogramming and support testing IM156 as a therapy for IPF and other fibrotic diseases. Significance Statement Fibrosing Interstitial Lung Diseases (FILD) have a poor prognosis and current anti-fibrotic treatments have significant limitations. This study demonstrates that attenuation of fibrogenic metabolic remodeling, by modulation of OXPHOS with IM156, prevents the myofibroblast phenotype/collagen deposition and is a potentially effective and translational anti-fibrotic strategy.

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) in the US accounting for about 22% of newly diagnosed cases of B-cell NHL. It is considered to be more aggressive and to have worse prognosis due to low response to existing treatments. KDM4 is a family of histone demethylases that can drive tumor growth by regulating transcription, cell cycle, and DNA replication/repair. Overexpression of KDM4 alters post-translational histone modification and is associated with many types of cancer, including DLBCL. TACH101 is a novel, potent small molecule inhibitor of KDM4 that is being developed for treatment of advanced cancers, including DLBCL. Methods: TACH101 was evaluated in vitro and in vivo using cancer cell lines and patient-derived organoid (PDO) and xenograft (PDX) models of DLBCL. Results: TACH101 is a reversible, α-ketoglutarate competitive, selective and potent inhibitor of KDM4 isoforms A-D with IC50 values < 0.100 μM for all four isoforms. An initial screen using a 301-cell line panel showed that DLBCL cell lines are sensitive to TACH101 (IC50 < 10 nM). In an additional panel of DLBCL cell lines, TACH101 inhibited proliferation in a dose-dependent manner in all DLBCL cell lines, independent of molecular subtype, with mean IC50’s of 0.03 ± 0.01 μM in ABC DLBCL cell lines (n=6); 0.02 ± 0.01 μM in GCB DLBCL cell lines (n=7) and 0.02 ± 0.01 μM in PMBL DLBCL cell lines (n=2). In OCI-LY19 DLBCL xenografts in vivo, TACH101 was well tolerated and inhibited tumor growth by 55% to 100%, depending on dosing regimen (either QD or BID following a 3 days on/4 days off schedule). In PK studies, TACH101 exhibited low clearance, moderate volume of distribution, and good oral bioavailability in mouse, rat, and dog. Moreover, treatment with TACH101 resulted in little or no inhibitory effects on CYP enzymes. Toxicity studies in rats and dogs identified potential target tissues and provided safety guidance for the use of TACH101 in human studies. Conclusions: The KDM4 inhibitor, TACH101, had compelling activity in preclinical DLBCL models, suggesting that TACH101 could be an effective therapy for DLBCL. Preparations to advance the drug into clinical trials are underway. Citation Format: Frank Perabo, Chandtip Chandhasin, Sanghee Yoo, Joselyn Del Rosario, Young K. Chen, Ellen Filvaroff, Jeffrey A. Stafford, Stephen Quake, Michael F. Clarke. TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase for the treatment of diffuse large B-cell lymphoma [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 3720.

132 Background: TACH101 is a novel, potent small molecule inhibitor of KDM4, a novel epigenetic target for cancer therapy. KDM4 is a family of histone lysine demethylases that, when overexpressed, drives key processes linked to cancer. Validation of KDM4 as a driver gene was confirmed across gastrointestinal tumor types including esophageal, colon and gastric cancers, and is associated with formation of aggressive tumors. Methods: TACH101 was evaluated in in vitro and in vivo studies including cell inhibition assays, patient-derived xenograft (PDX) and organoid models, and bioinformatics analyses studies. Results: In vitro, TACH101 treatment potently inhibited cell proliferation in cell lines and organoid models representing esophageal, CRC, and gastric cancers. TACH101 induced apoptosis in human CRC (HT-29) and esophageal (KYSE-150) cancer cell lines (EC50s 0.033 - 0.092 µM). Further evaluation using a panel of > 300 cell lines from different tumor types showed potent activity of TACH101 against gastric cancer and CRC. In gastric cancer, 2D cell viability inhibition assays conducted on a panel of 11 gastric cancer cell lines showed 9/11 (82%) were sensitive to TACH101 treatment (IC50 0.004 - 0.072 µM); in PDX models, 4/5 (80%) were sensitive to TACH101 treatment (IC50 0.007 - 0.039 µM). In CRC, bioinformatics analysis indicated increased TACH101 sensitivity in cell lines with MSI-H status (IC50 1-150 nM). Sensitivity of MSI-H CRC to TACH101 was further confirmed in a panel of 14 CRC PDX models and 7 CRC organoid models in culture-based viability inhibition assays. In PDX models, 5/5 (100%) characterized as MSI were sensitive to TACH101 treatment (IC50 0.001 - 0.014 µM), whereas 4/8 (50%) characterized as MSS were sensitive to TACH101 (IC50 0.003 - 0.270 µM). In patient derived CRC organoid models, 3/3 (100%) characterized as MSI were sensitive to TACH101 treatment (IC50 0.022 - 0.149 µM) whereas 0/3 (0%) characterized as MSS were sensitive (IC50 > 10 µM). In vivo, TACH101 triggered effective tumor control (≥70%) in xenograft models of CRC (SU60), esophageal (KYSE-150) and gastric (GXA-3036) cancers. Pharmacologic studies showed TACH101 demonstrated favorable cell permeability, good oral bioavailability, and high metabolic stability. Conclusions: Preclinical work on TACH101 KDM4 inhibitor demonstrates compelling data and applicability as a potential therapy for gastrointestinal cancers. Preparations to advance TACH101 into clinical trials are underway.

This study analyzes how the shacho-kai (presidents’ meeting) have contributed to the competitiveness of the Japanese keiretsu network. shacho-kai had been touched upon by various literatures as one of the important elements of the keiretsu, but there had been no attempt to analyze the shacho-kai in detail with comprehensive framework. Thus, this study analyzes the shacho-kai of Mitsubishi group by applying the ABCD model, which is a framework to explain competitiveness or the success factors and compare with the case of sajangdan-hoeui of Samsung, South Korea. Samsung’s sajangdan-hoeui has shown superiority in speed and diligence whereas shacho-kai of Mitsubishi was better in synergy creation and goal orientation factors.

Targeting hair follicle regeneration has been investigated for the treatment of hair loss, and fundamental studies investigating stem cells and their niche have been described. However, knowledge of stem cell metabolism and the specific regulation of bioenergetics during the hair regeneration process is currently insufficient. Here, we report the hair regrowth-promoting effect of a newly synthesized novel small molecule, IM176OUT05 (IM), which activates stem cell metabolism. IM facilitated stemness induction and maintenance during an induced pluripotent stem cell generation process. IM treatment mildly inhibited mitochondrial oxidative phosphorylation and concurrently increased glycolysis, which accelerated stemness induction during the early phase of reprogramming. More importantly, the topical application of IM accelerated hair follicle regeneration by stimulating the progression of the hair follicle cycle to the anagen phase and increased the hair follicle number in mice. Furthermore, the stem cell population with a glycolytic metabotype appeared slightly earlier in the IM-treated mice. Stem cell and niche signaling involved in the hair regeneration process was also activated by the IM treatment during the early phase of hair follicle regeneration. Overall, these results show that the novel small molecule IM promotes tissue regeneration, specifically in hair regrowth, by restructuring the metabolic configuration of stem cells., Stem cells: A remedy to revitalize the roots A compound that establishes metabolic conditions favorable for sustaining stem cells may also offer a safe drug for promoting hair regrowth. Drugs that inhibit mitochondrial activity help lock stem cells into a pluripotent state that allows them to actively divide and repair various tissues, but many of these drugs are toxic. Researchers led by Myung Jin Son of the Korea Research Institute of Bioscience and Biotechnology, Daejeon and ImmunoMet, USA have identified a new compound that safely achieves the same effect. This potential drug helped promote stemness in diverse stem cell types, including the highly proliferative cells that comprise hair follicles. Topical application proved more effective at promoting hair regrowth in female mice than the baldness drug minoxidil, and matched its performance in males, demonstrating its potency as a stem cell modulator.

Background: TACH101 is a novel, selective and potent inhibitor of KDM4 in development for advanced cancers. KDM4 plays a key role in epigenetic regulation by removing methyl marks on histone H3K9 and H3K36. Overexpression of KDM4 leads to dysregulation of transcription, cell cycle, and DNA replication/repair processes and has been associated with many cancer types. Methods: Candidate pharmacodynamic (PD) and sensitivity biomarkers for TACH101 were evaluated in vitro and in vivo using cancer cell lines, mouse xenograft models, gene microarrays, ChIP-seq, and ChIP-qPCR. Results: Evaluation of differential gene expression from tumor tissue identified several candidates as potential PD markers for TACH101 activity. In particular, direct binding of KDM4 to Protein Phosphatase 1 Regulatory subunit 10 (PPP1R10 or PNUTS) was confirmed by ChIP-seq in cell lines from esophageal (KYSE-150), breast (MDA-MB-231) and lymphoma (OCI-Ly19) cancers. TACH101 treatment caused 86% repression of PNUTS mRNA, as well as a 51% increase in H3K9me3, a mark of repressed transcription. A 78% decrease in H3K36me3 at the PNUTS gene was also observed. PNUTS as a target of KDM4 was validated via ChIP-qPCR in xenograft tumors of KYSE-150, OCI-Ly19 and SU-60 (colorectal). In vivo, maximum inhibition of PNUTS was reached at 8 hours post TACH101 administration, when TACH101 concentration was 100-200 nM in tumors. In addition, extensive bioinformatics analysis using >300 cancer cell lines showed that cell lines with MSI-high (MSI-H) status tended to be more sensitive to TACH101 in vitro. This association was found with other markers of MSI-H status such as MMR gene mutations, MLH1 methylation status, and overall mutation frequency. To further test this association, TACH101 was evaluated in a panel of patient-derived xenograft (PDX) and organoid models. The results showed a strong correlation of TACH101 sensitivity with MSI-H status (IC50 ranges 1-150 nM). Conclusions: Candidate PD biomarkers of TACH101 activity for clinical evaluation have been identified and further studies are ongoing to explore current and additional markers. In addition, MSI-H status was associated with increased sensitivity to TACH101, which may be useful for enriching for select patient populations in clinical studies. Citation Format: Frank Perabo, Sanghee Yoo, Chandtip Chandhasin, Joselyn Del Rosario, Young K. Chen, Ellen Filvaroff, Jeffrey A. Stafford, Stephen Quake, Michael F. Clarke. Identification of pharmacodynamic and sensitivity biomarkers for TACH101, a pan-inhibitor of KDM4 histone lysine demethylase [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 P086.

Background: Alterations in epigenetic control can cause defective post-translational histone modification and dysregulation of gene activity leading to cancer development. Overexpression of KDM4, a demethylase of histone lysine 9 methyl 2/3 and lysine 36 methyl 2/3 (H3K9me2/3 and H3K36me2/3), is documented in a variety of cancers and is linked to aggressive disease and poor clinical outcomes. TACH101 is a novel first-in-class, selective and potent pan KDM4 inhibitor with favorable pharmacologic properties. Methods: TACH101 was evaluated using in vitro and in vivo studies including cell-proliferation assays in multiple cancer cell lines and patient-derived organoid models, apoptotic and cell cycle analyses, pharmacology studies in various animal species, and efficacy studies in xenograft tumor models. Results: Inhibition mechanism studies demonstrated TACH101 to be a reversible, α-ketoglutarate competitive, selective and potent inhibitor of KDM4 isoforms A-D with IC50 values less than 0.100 μM for all four isoforms. In vitro, TACH101 demonstrated potent increase of H3K36me3 levels (EC50 Conclusions: TACH101 is a potent pan inhibitor of KDM4 that is suggestive of broad potential in cancer treatment. Further preclinical evaluation is ongoing to advance the molecule into clinical trials. Citation Format: Sanghee Yoo, Chandtip Chandhasin, Joselyn R. Del Rosario, Young K. Chen, Jeff Stafford, Stephen Quake, Frank Perabo, Michael F. Clarke. TACH101, a first-in-class pan inhibitor of KDM4 histone lysine demethylases [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 2128.

Cancer cells display metabolic plasticity to survive metabolic and energetic stresses in the tumor microenvironment, prompting the need for tools to target tumor metabolism. Cellular adaptation to energetic stress is coordinated in part by signaling through the Liver Kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Reducing LKB1-AMPK signaling exposes metabolic vulnerabilities in tumor cells with potential for therapeutic targeting. Here we describe that miRNA-mediated silencing of LKB1 (mediated by the oncogenic miRNA clustermiR-17∼92) confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and novel (IM156) biguanides, we demonstrate thatMyc+lymphoma cells with elevatedmiR-17∼92expression display increased sensitivity to biguanide treatment both in cell viability assaysin vitroand tumor growth assaysin vivo. This increased biguanide sensitivity is driven bymiR-17-dependent silencing of LKB1, which results in reduced AMPK activation in response to bioenergetic stress. Mechanistically, biguanide treatment inhibits TCA cycle metabolism and mitochondrial respiration inmiR-17∼92-expressing tumor cells, targeting their metabolic vulnerability. Finally, we demonstrate a direct correlation betweenmiR-17∼92expression and biguanide sensitivity in human cancer cells. Our results identifymiR-17∼92expression as a potential biomarker for biguanide sensitivity in hematological malignancies and solid tumors.One Sentence SummarymiR-17∼92 expression inMyc+tumors sensitizes cancer cells to biguanide treatment by disrupting bioenergetic stability in lymphoma cells.

Summary Cancer cells display metabolic plasticity to survive stresses in the tumor microenvironment. Cellular adaptation to energetic stress is coordinated in part by signaling through the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Here, we demonstrate that miRNA-mediated silencing of LKB1 confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and newly developed (IM156) biguanides, we demonstrate that elevated miR-17∼92 expression in Myc+ lymphoma cells promotes increased apoptosis to biguanide treatment in vitro and in vivo. This effect is driven by the miR-17-dependent silencing of LKB1, which reduces AMPK activation in response to complex I inhibition. Mechanistically, biguanide treatment induces metabolic stress in Myc+ lymphoma cells by inhibiting TCA cycle metabolism and mitochondrial respiration, exposing metabolic vulnerability. Finally, we demonstrate a direct correlation between miR-17∼92 expression and biguanide sensitivity in human cancer cells. Our results identify miR-17∼92 expression as a potential biomarker for biguanide sensitivity in malignancies., Graphical Abstract, Highlights IM156 is a newly developed biguanide with more potency than phenformin IM156 inhibits mitochondrial respiration to cause bioenergetic stress in lymphoma miR-17-dependent silencing of LKB1 reduces adaptation to bioenergetic stress miR-17∼92 expression correlates with biguanide sensitivity in human cancer cells, Izreig et al. show that miR-17∼92 expression in cancer cells sensitizes them to biguanide treatment by disrupting bioenergetic stability. These data implicate miR-17∼92 expression as a potential biomarker for biguanide sensitivity in hematological malignancies and possibly solid tumors.

e15067 Background: The histone Lysine (K) Demethylase 4 family, KDM4 family, is involved in nuclear functions such as programming development, activation or repression of transcription, timing and control of the cell cycle, and initiating DNA replication and repair through chromatin remodeling. Overexpression of KDM4 leading to mistakes in post-translational histone modification has been associated with many cancer types and is being investigated as a potential therapeutic drug target. TACH101 is a novel potent and small molecule inhibitor of the KDM4 family. It demonstrated strong efficacy in multiple cancer types in vitro and in vivo and favorable pharmacokinetics (PK) and safety profiles in preclinical models. Methods: The target interaction properties of TACH101 were evaluated using purified enzymes for biochemical, biophysical, and inhibition mechanism studies. For PK parameter measurements, Sprague-Dawley rats and Beagle dogs were used for intravenous or oral administration of TACH101. Hepatocytes or liver microsomes were used to measure cytochrome P450 (CYP) inhibition and induction studies. Cell lines expressing cardiac channels were used for radioligand displacement assays and patch clamping. Results: Biochemical, biophysical, and inhibition mechanism studies using purified enzymes showed TACH101 is a reversible, alpha-KG (co-factor) competitive, and slow-binding inhibitor with IC50 values less than 100 nM. Pharmacologic studies showed plasma protein binding of TACH101 to be ≥99% bound in mouse, rat, dog, and human. TACH101 appeared to be selective in off-target panel assays of receptors, ion channels and transporters. TACH101 did not show any significant binding displacement activity against cardiac ion channels or any effect on hERG in CHO cells using manual patch clamp techniques. In rats and dogs, TACH101 exhibited low systemic clearance, a moderate volume of distribution in rats and dogs, linear exposure profiles with a terminal half-life (̃ 6-hour) and high bioavailability (46-100%). In repeated dose studies in rats and dogs, mean Cmax and AUC values increased in a dose-related manner over the dose range evaluated. There was no significant impact of food on systemic exposure in dogs. TACH101 did not show inhibition or induction of CYP enzymes tested. Conclusions: TACH101 is a potent KDM4 specific inhibitor without significant off-target activity in in vitro and in vivo studies. It did not show inhibition or induction of CYP enzymes suggesting low probability of CYP mediated drug-drug interaction. The exposure from oral administration in rats and dogs was dose proportional and was not significantly affected by food intake in dogs. Given the favorable target activity, PK properties and safety profile, a Phase 1 study is being planned to advance TACH101 for patients with advanced cancer.

3105 Background: The KDM4 family of histone lysine demethylases consists of four main isoforms (KDM4A, B, C, D), all of which have been identified as key oncogenic drivers. They function as epigenetic regulators and control transitions between transcriptionally silent and active chromatin states via removal of methyl marks on histone H3K9 and histone H3K36. KDM4 isoforms play an important role in the epigenetic dysregulation in various cancers and is linked to more aggressive disease and poorer clinical outcomes. Functional redundancy and cross-activity have been observed across KDM4 family members, thus, selective inhibition of one isoform appears to not be effective. TACH101 is a novel, first-in-class pan inhibitor of KDM4 that simultaneously targets multiple isoforms of KDM4. Here we present data that show TACH101 has promising pre-clinical and pharmacologic properties as a cancer therapeutic. Methods: TACH101 was evaluated in in vitro and in vivo studies including cell-proliferation assays in multiple cancer cell lines, apoptotic and cell cycle analyses, and efficacy studies in various xenograft tumor models and patient-derived organoid models. Results: In vitro, TACH101 was broadly effective in killing 67% (200 out of 300) of cancer cell lines screened. TACH101 demonstrated potent increase of H3K36me3 levels (EC50 < 0.001 mM, HTRF) in KYSE-150 cell line engineered to overexpress KDM4C and potent anti-proliferative activity in multiple cell lines in OncoPanel. TACH101 treatment increased cancer cell population in S-phase in multiple cancer cell lines indicating cell-cycle arrest. TACH101 induced apoptosis in human colorectal (HT-29), esophageal (KYSE-150), and triple negative breast cancer (MDA-MB-231) cell lines with EC50s ranging from 0.033-0.092 µM. In vivo, TACH101 triggered effective tumor control in xenograft models including colorectal, esophageal, gastric, breast, and lymphoma with tumor growth inhibition of up to 100%. Further evaluation using a panel of patient-derived colorectal models and patient-derived organoids showed a strong correlation of TACH101 sensitivity with MSI-H status (IC50 ranges 1-150 nM). TACH101 also reduced tumorigenic potential by 4.4-fold as determined by FACS analysis using sorted CD44High EpCAM+ population in Limiting Dilution Assays in vivo, suggesting that reduction of cancer stem cells by TACH101 may be effective in therapy-resistant settings. Pharmacologic studies showed TACH101 demonstrated favorable cell permeability, good oral bioavailability, and high metabolic stability. Conclusions: Extensive preclinical work on TACH101 KDM4 inhibitor shows compelling data and broad applicability as a potential anti-cancer agent. Further evaluation is ongoing to advance the molecule into clinical trials.

3590 Background: IM156, a novel oral potent biguanide OXPHOS inhibitor of Protein Complex 1 (PC1) of the mitochondrial electron transport chain, causes AMPK phosphorylation, the downstream effects of which are detrimental to OXPHOS-dependent cancer cells prone to energy stress. Preclinical experiments with IM156 demonstrated activity in solid tumor and hematologic malignancy models as a single-agent and in combinations. Methods: This was an open label, first-in-human, multi-center, dose-escalation study ( NCT03272256 ) using a 3+3 design. The primary endpoint was to determine the maximum tolerated dose and/or recommended Phase 2 dose (RP2D) based on dose limiting toxicities (DLT), safety and tolerability. Secondary endpoints included pharmacokinetics (PK), pharmacodynamics (PD) and preliminary signals of efficacy. Eligible patients were adults with advanced solid tumors refractory to standard therapies with ECOG Performance Status max and AUC0-last reaching the expected efficacious range. PD demonstrated a decrease in tumor growth rate in 3 patients (1,200 mg QOD: N = 2; 800 mg QD: N = 1), and a decrease in VEGF and tumor markers in a patient with gastric cancer with neuroendocrine differentiation treated at 800 mg QD who remains on study in Cycle 11. Best response was stable disease in 7 (32%) patients. Conclusions: IM156 is the first PC1 OXPHOS inhibitor to have been successfully tested in patients with cancer with the identification of a RP2D. It was well tolerated at dose levels active in preclinical models, and demonstrated modest clinical activity in an unselected population of patients. Subsequent development will focus on OXPHOS-dependent tumors and in combinations with agents in which OXPHOS metabolism is a mechanism of resistance. Clinical trial information: NCT03272256 .

Calcitonin gene-related peptide (CGRP) has been implicated in acute migraine pathogenesis. In an effort to identify novel CGRP receptor antagonists for the treatment of migraine, we have discovered thiazolidinone 49, a potent (Ki=30 pM, IC50=1 nM), orally bioavailable, CNS-penetrant CGRP antagonist with good pharmacokinetic properties.

TPS2620Background: IM156 is a novel oral agent with a biguanide structure, which has anticancer activity through AMPK activation and reduction of oxidative phosphorylation. Inhibition of oxidative ...

Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the β-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.

Pseudotyped retroviral vectors combine the advantages of broad host range, high expression, stable chromosomal integration, and ease of preparation. These vectors greatly facilitate delivery into mammalian cells of sequences encoding individual peptide inhibitors—including those with therapeutic utility—and inhibitor libraries. However, retroviral vectors vary in behavior, particularly with respect to expression levels in different cell lines. Expression level is especially important in transdominant experiments because the concentration of an inhibitor (for example, an expressed peptide) is one of the key determinants in the degree of complex formation between the inhibitor and its target. Thus, inhibitor concentration should have an impact on the expressivity and/or penetrance of an induced phenotype. Here, we compare several retroviral vectors and human cell lines for relative expression levels using a green fluorescent protein reporter. We show for a subset of these lines that cellular protein concentrations produced by single-copy vectors range up to about 2 μM. We also examine other variables that contribute to expression level, such as the nature of the expressed protein's carboxy terminus. Finally, we test the effect of increased concentration on phenotype with a nine-amino-acid peptide derived from the human papilloma virus protein E7 which overcomes E7-mediated cell growth.

The human immunodeficiency virus type I (HIV-1) capsid protein is initially synthesized as the central domain of the Gag polyprotein, and is subsequently proteolytically processed into a discrete 231-amino-acid protein that forms the distinctive conical core of the mature virus. The crystal structures of two proteins that span the C-terminal domain of the capsid are reported here: one encompassing residues 146-231 (CA146-231) and the other extending to include the 14-residue p2 domain of Gag (CA146-p2). The isomorphous CA146-231 and CA146-p2 structures were determined by molecular replacement and have been refined at 2.6 A resolution to R factors of 22.3 and 20.7% (Rfree = 28.1 and 27.5%), respectively. The ordered domains comprise residues 148-219 for CA146-231 and 148-218 for CA146-p2, and their refined structures are essentially identical. The proteins are composed of a 310 helix followed by an extended strand and four alpha-helices. A crystallographic twofold generates a dimer that is stabilized by parallel packing of an alpha-helix 2 across the dimer interface and by packing of the 310 helix into a groove created by alpha-helices 2 and 3 of the partner molecule. CA146-231 and CA146-p2 dimerize with the full affinity of the intact capsid protein, and their structures therefore reveal the essential dimer interface of the HIV-1 capsid.

The HIV-1 capsid protein (CA) makes an essential interaction with the human peptidyl prolyl isomerase, cyclophilin A (CypA), that results in packaging of CypA into the virion at a CA to CypA stoichiometry of ∼10:1. The 231 amino acid residue capsid protein is composed of an amino-terminal CypA binding domain (1 to ∼151; CA151) and a carboxyl-terminal dimerization domain (∼151 to 231). We find that CypA binds dimeric CA and monomeric CA151 with identical intrinsic affinities (Kd=16(±4) μM). This result demonstrates that capsid dimerization and cyclophilin A binding are not thermodynamically coupled and suggests that the substoichiometric ratio of CypA in the HIV-1 virion results from the intrinsic stability of the CA/CypA complex. In the known co-crystal structure of the CA151/CypA complex, CypA binding is mediated exclusively by an exposed capsid loop that spans residues Pro85 to Pro93. The energetic contributions to CypA binding were quantified for each residue in this loop, and the results demonstrate that the Gly89-Pro90 dipeptide is the primary cyclophilin A recognition motif, with Pro85, Val86, His87, Ala88, and Pro93 also making energetically favorable contacts. These studies reveal that the active site of CypA, which can catalyze the isomerization of proline residues in vitro, also functions as a sequence-specific, protein-binding motif in HIV-1 replication.

The HIV-1 capsid protein forms the conical core structure at the center of the mature virion. Capsid also binds the human peptidyl prolyl isomerase, cyclophilin A, thereby packaging the enzyme into the virion. Cyclophilin A subsequently performs an essential function in HIV-1 replication, possibly helping to disassemble the capsid core upon infection. We report the 2.36 A crystal structure of the N-terminal domain of HIV-1 capsid (residues 1–151) in complex with human cyclophilin A. A single exposed capsid loop (residues 85–93) binds in the enzyme's active site, and Pro-90 adopts an unprecedented trans conformation. The structure suggests how cyclophilin A can act as a sequence-specific binding protein and a nonspecific prolyl isomerase. In the crystal lattice, capsid molecules assemble into continuous planar strips. Side by side association of these strips may allow capsid to form the surface of the viral core. Cyclophilin A could then function by weakening the association between capsid strips, thereby promoting disassembly of the viral core.

Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the beta-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.

The cellular protein, cyclophilin A (CypA), is incorporated into the virion of the type 1 human immunodeficiency virus (HIV-1) via a direct interaction with the capsid domain of the viral Gag polyprotein. We demonstrate that the capsid sequence 87His-Ala-Gly-Pro-Ile-Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site and present an X-ray crystal structure of the CypA/HAGPIA complex. In contrast to the cis prolines observed in all previously reported structures of CypA complexed with model peptides, the proline in this peptide, Pro 90, binds the cyclophilin A active site in a trans conformation. We also report the crystal structure of a complex between CypA and the hexapeptide HVGPIA, which also maintains the trans conformation. Comparison with the recently determined structures of CypA in complexes with larger fragments of the HIV-1 capsid protein demonstrates that CypA recognition of these hexapeptides involves contacts with peptide residues Ala(Val) 88, Gly 89, and Pro 90, and is independent of the context of longer sequences.

The three-dimensional structure of the amino-terminal core domain (residues 1 through 151) of the human immunodeficiency virus-type 1 (HIV-1) capsid protein has been solved by multidimensional heteronuclear magnetic resonance spectroscopy. The structure is unlike those of previously characterized viral coat proteins and is composed of seven alpha helices, two beta hairpins, and an exposed partially ordered loop. The domain is shaped like an arrowhead, with the beta hairpins and loop exposed at the trailing edge and the carboxyl-terminal helix projecting from the tip. The proline residue Pro1 forms a salt bridge with a conserved, buried aspartate residue (Asp51), which suggests that the amino terminus of the protein rearranges upon proteolytic maturation. The binding site for cyclophilin A, a cellular rotamase that is packaged into the HIV-1 virion, is located on the exposed loop and encompasses the essential proline residue Pro90. In the free monomeric domain, Pro90 adopts kinetically trapped cis and trans conformations, raising the possibility that cyclophilin A catalyzes interconversion of the cis- and trans-Pro90 loop structures.

Summary Cancer cells display metabolic plasticity to survive stresses in the tumor microenvironment. Cellular adaptation to energetic stress is coordinated in part by signaling through the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Here, we demonstrate that miRNA-mediated silencing of LKB1 confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and newly developed (IM156) biguanides, we demonstrate that elevated miR-17∼92 expression in Myc+ lymphoma cells promotes increased apoptosis to biguanide treatment in vitro and in vivo. This effect is driven by the miR-17-dependent silencing of LKB1, which reduces AMPK activation in response to complex I inhibition. Mechanistically, biguanide treatment induces metabolic stress in Myc+ lymphoma cells by inhibiting TCA cycle metabolism and mitochondrial respiration, exposing metabolic vulnerability. Finally, we demonstrate a direct correlation between miR-17∼92 expression and biguanide sensitivity in human cancer cells. Our results identify miR-17∼92 expression as a potential biomarker for biguanide sensitivity in malignancies.