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Supplementary methods, data and Figures

After significant effort over the last 30 years, antibody–drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

Supplementary Figure S1. TH-302 has potent antitumor activity in a 3D in vitro co-culture system of leukemia cells and bone marrow-derived mesenchymal stromal cells that recapitulates the multidimensional BM niche Supplementary Figure S2. Hypoxia is present in spheroids as indicated by PIMO positive staining in spheroids incubated with PIMO for 3hr before harvesting Supplementary figure S3. In vitro synergistic activity of TH-302 in combination with chemotherapy and demethylating agents Supplementary Figure S4. TH-302 has anti-leukemia activity in an in vivo primary AML xenograft murine model. Supplementary figure S5. In vitro synergistic activity of TH-302 in combination with sorafenib Supplementary figure S6 A, B. Assessment of BM hypoxia in AML/ETO bearing mice treated with TH-302 or chemotherapy

Supplemental Methods and Tables 1) mRNA Hybridization and Gene-expression Profiling. 2) Gene expression analysis by Nanostring technology. 3) Hyperpolarized Magnetic Resonance Spectroscopy. 4) In Vitro 1-13C Pyruvic Acid Feeding Studies and High Resolution Nuclear Magnetic Resonance (NMR) Experiments 5) Three-dimensional spheroids 6) Murine tumor models 7) Immunohistochemistry Supplementary Table S1. Cell line characteristics Supplementary Table S2. Patient characteristics Supplemental Table S3. List of upregulated DEPs in hypoxia. Supplemental Table S4. 1-13C pyruvate uptake and 1-13C lactate production in cell culture. Supplemental Table S5. TH-302 cytotoxic activity against leukemia cell lines

Purpose: To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models.Experimental Design: Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models.Results: Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a “hypoxia index” compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo. In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells.Conclusions: These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins. Clin Cancer Res; 22(7); 1687–98. ©2015 AACR.

Eranthis Salisb. (Ranunculaceae) is a herbaceous plant genus, including few species disjunctively distributed throughout the temperate zone from Southeastern Europe to Eastern Asia. Until recently, only Eranthis sibirica DC. was known in South Siberia, being considered endemic and tertiary relict. Not long ago, Eranthis tanhoensis Erst was also described in Siberia. We report here a reconstruction of the phylogenetic relationships between the Siberian Eranthis species based on nuclear (ITS) and plastid (trnL + trnL-trnF + trnH-psbA) DNA. The phylogeographic structure of Siberian Eranthis is distinguished by the presence of the two “eastern” and “western” supergroups, which most likely formed as a result of disjunction caused by active mountain uplifts during the late Neogene–early Quaternary and subsequent progressive Pleistocene cooling. The eastern supergroup combines lineage I, containing populations from the eastern Khamar-Daban Ridge, the Eastern Sayan Mountains, and the Tannu-Ola Ridge, and lineage II containing western Khamar-Daban populations. The western supergroup includes only lineage III, containing Western Sayan populations. Our data clearly show that E. tanhoensis is nested in the E. sibirica clade, thereby indicating that its description as a separate species is unjustified, as it compromises the monophyletic status of E. sibirica. Therefore, we suggest here to consider E. tanhoensis as a synonym of E. sibirica.

Tuberculosis (TB) has surpassed HIV to become the leading infectious killer of adults globally, causing almost 2 million deaths annually. Although this airborne disease has been treatable since 1948, global rates of TB have dropped less than two percent per year; an estimated 10 million incident cases continue to occur annually, including one million in children. While transmission of active disease is an important driver of the epidemic, the seedbed that feeds the epidemic is the more than two billion people estimated to have TB infection, five to ten percent of whom will progress to active disease during their lifetime. While any successful strategy aimed at TB elimination needs to address this reservoir of TB infection worldwide, much remains to be understood about host and pathogen factors that can be used to identify increased risk for progression to disease, and intervened upon to prevent progression from occurring. The Division of AIDS of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA, and the Harvard Medical School Center for Global Health Delivery-Dubai convened a group of scientists and stakeholders on September 28 and 29, 2017, to address knowledge gaps that affect our ability to rapidly find and treat individuals infected with Mycobacterium tuberculosis who are most likely to progress to active disease. The meeting identified a number of efforts underway to address this important gap in the collective ability to stop the global TB epidemic. Here, we review and outline the priority areas for research, diagnosis and treatment of TB infection that emerged from the meeting (Table 1), building on recent reviews in this area.

Pyrrolobenzodiazepine (PBD) dimers are well-known highly potent antibody drug conjugate (ADC) payloads. The corresponding PBD monomers, in contrast, have received much less attention from the ADC community. We prepared several novel polyamide-linked PBD monomers and evaluated their utility as ADC payloads. The unconjugated polyamide-PBD hybrids exhibited potent antiproliferative activity (IC50 range: 10

STING pathway agonism has emerged as a potential therapeutic strategy to stimulate anti-tumor immune responses. We have previously shown that tumor cell-targeted antibody-drug conjugates (ADCs) carrying a novel STING agonist induce anti-tumor activity without causing substantial elevations in systemic cytokine levels, thus suggesting a therapeutic advantage of STING agonist ADCs relative to unconjugated agonists. ADCs constitute a proven therapeutic modality that is ideally suited to enable systemic administration and delivery of the conjugated drug to desired cell types within the tumor microenvironment. In addition to delivering STING agonist into the antigen-expressing tumor cells, antigen-bound ADCs deliver STING agonist to tumor-resident myeloid cells through Fcγ receptor (FcγR)-mediated internalization. In this study we investigated the mechanism of FcγR-mediated internalization of the tumor cell-targeted STING-agonist ADCs into myeloid cells and the nature of the subsequent STING pathway activation. We developed flow cytometry-based assays to determine the changes in FcγRI, FcγRII, and FcγRIII cell surface detection levels in the presence of ADCs specifically designed to be either proficient or deficient in FcγR-binding. Combined with functional assays based on co-culture of cancer cells and FcγRI knock out myeloid cells, we identified FcγRI as the major Fcγ receptor that mediates target-bound ADC internalization into myeloid cells in vitro. Even though FcγRI is expressed only by a subset of CD11b+ myeloid cells, tumor cell-targeted ADCs induce greater production of interferons and other cytokines and more potent cancer cell killing than CD11b-targeted-ADCs, which deliver STING agonist into FcγRI- (non-productive) as well as FcγRI+ (productive) myeloid cells. Finally, we demonstrate that myeloid cells within dissociated primary human tumors from multiple donors express FcγRI and are capable of tumor cell killing in response to tumor cell-targeted STING agonist ADCs in vitro. In summary, our data indicate that the ADC-mediated delivery of a STING agonist specifically into FcγRI-expressing myeloid cells efficiently activates innate immune responses in the most relevant immune cell types within the tumor microenvironment. Citation Format: Naniye Malli Cetinbas, Kalli C. Catcott, Travis Monnell, Jahna Soomer-James, Keith Bentley, Susan Clardy, Bingfan Du, Eoin Kelleher, Marina Protopopova, Cheri Stevenson, Joshua D. Thomas, Alex Uttard, Dorin Toader, Jeremy Duvall, Raghida Bukhalid, Marc Damelin, Timothy B. Lowinger. Tumor cell-targeted STING-agonist antibody-drug conjugates achieve potent anti-tumor activity by delivering STING agonist specifically to tumor cells andFcγRI-expressing subset of myeloid cells [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 2114.

We present here a novel therapeutic agent, XMT-2056, that results in robust anti-tumor activity mediated by an immune response through targeted delivery of a STING agonist to the tumor microenvironment. By leveraging an antibody-drug conjugate (ADC) strategy, systemic administration of a STING agonist with tumor-targeted delivery can be achieved, potentially overcoming limitations of either intratumoral or intravenous administrations of unconjugated, small molecule STING agonists. XMT-2056 was generated through conjugation of Immunosynthen, a platform that employs a novel STING agonist payload specifically designed for ADCs, to HT-19, a HER2-targeting antibody which binds to a novel epitope and does not compete for binding with either trastuzumab or pertuzumab. Initial results showed XMT-2056 has target-dependent anti-tumor activity in vivo and is well tolerated in non-human primates at significantly higher exposure levels than those required for anti-tumor activity. To evaluate the impact of HER2 expression level on the activity of XMT-2056, in vivo studies in gastric and breast cancer models with varying HER2 expression levels were conducted, and XMT-2056 showed potent anti-tumor activity in a dose dependent and target dependent manner including in models with very low expression of HER2. Because the antibody employed in XMT-2056 does not compete for binding with trastuzumab or pertuzumab, we hypothesized that there could be benefit in combining with such approved HER2-targeted therapies. This advantage was demonstrated in vivo as the combination of XMT-2056 and trastuzumab or pertuzumab showed greater anti-tumor activity compared to the administration of either agent alone. Further efforts to elucidate the mechanism(s) of the observed benefit of these combinations will be discussed. Given the innate immune activation by XMT-2056, there is also a strong rationale for combination with immune checkpoint inhibitors. To this end, administration of an XMT-2056 surrogate ADC in combination with an anti-PD1 agent improved anti-tumor activity in a ratHER2-engineered EMT-6 syngeneic mouse model. Together these data support the potential of XMT-2056 both as a monotherapy and in combination with other HER2 targeted agents as well as checkpoint inhibitors. Citation Format: Jeremy R. Duvall, Raghida A. Bukhalid, Naniye M. Cetinbas, Kalli C. Catcott, Kelly Lancaster, Keith W. Bentley, Suzanna Clark, Susan Clardy, Scott D. Collins, Anouk Dirksen, Elizabeth Ditty, Bingfan Du, Eugene W. Kelleher, Travis Monnell, Marina Protopopova, Caitlin Routhier, Cheri Stevenson, Elena Ter-Ovanesyan, Joshua D. Thomas, Alex Uttard, Jason Wang, Phonphimon Wongthida, Ling Xu, Annika Yau, Jeffrey Zurita, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-2056, a HER2-targeted Immunosynthen STING-agonist antibody-drug conjugate, binds a novel epitope of HER2 and shows increased anti-tumor activity in combination with trastuzumab and pertuzumab [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 3503.

After significant effort over the last 30 years, antibody–drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

BackgroundThe tumor microenvironment is a complex, multicellular system, composed not only of malignant cancer cells but also of a diversity of stromal cells including vascular cells, immune cells, and fibroblasts that support tumorigenesis. Antigens expressed on these cells tend to be widely expressed across a range of malignancies, presenting unique opportunities for development of anti-cancer therapies.MethodsWe have previously demonstrated that STING-agonist antibody-drug conjugates (Immunosynthen ADCs) targeting tumor cell antigens induce target-dependent anti-tumor immune responses in vitro and in vivo. To that effect, we hypothesized that Immunosynthen ADCs targeting tumor-associated antigens would coordinate immune-mediated killing of cancer cells not expressing the tumor-associated antigens (antigen-negative cancer cells) and induce anti-tumor activity.ResultsHerein, we demonstrate that targeting tumor-associated antigens with STING-agonist ADCs activate the STING pathway in immune cells via Fcγ receptor-mediated uptake. In addition, due to the intrinsic ability of certain tumor-associated cells to activate the STING pathway, STING-agonist ADCs targeting those cells can induce STING signaling in both the targeted cells and the immune cells, which constitutes a therapeutic advantage of ADCs that activate the STING pathway. In triple co-cultures of antigen-positive tumor-associated cells, antigen-negative cancer cells, and immune cells, the STING-agonist ADC specifically induced potent cell killing of the antigen-negative cancer cells with minimal impact on the immune and tumor-associated cells, thus representing a non-traditional, yet highly effective mechanism of ADC targeting. In vivo efficacy studies showed that STING-agonist ADCs developed for two tumor-associated antigens induced complete, sustained tumor regressions in syngeneic tumor models and exhibited immunological memory after rechallenge. CD8+ T cells contributed to the anti-tumor activity of the STING-agonist ADCs.ConclusionsIn summary, Immunosynthen STING-agonist ADCs targeting tumor-associated antigens represent a novel approach for ADC-mediated cancer immunotherapy and enable the multifaceted activation of the STING pathway in a tumor-targeted manner beyond tumor antigens.

STING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an innate anti-tumor immune response. However, the systemic administration of a free STING agonist may be limited by toxicity, and broad biodistribution may not be ideal. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that enables tumor-targeted delivery and thus is ideally suited to systemic administration with reduced toxicity. To develop an optimized STING-agonist ADC platform, we designed a novel STING-agonist specifically tailored for use in an ADC. Determination of the co-crystal structure confirmed that the agonist binds to the closed, or ‘active', conformation of the STING homodimer. The resulting Immunosynthen platform, which was developed specifically for the selected STING agonist payload, was used to generate XMT-2056, a tumor antigen-targeted STING-agonist ADC with excellent drug-like properties and >100-fold increased potency as compared to the free STING-agonist payload. In mice, XMT-2056 induced robust anti-tumor immune activity, with only minimal increases in systemic cytokine levels, and exhibited significant benefit over the benchmark free STING-agonist payload in both regards. Additionally, in vitro and in vivo studies demonstrate that XMT-2056 is able to activate the STING pathway in both tumor-resident immune cells and tumor cells, offering a potential advantage over other innate immune activating pathways. XMT-2056 was well-tolerated in non-human primates at significantly higher exposure levels than those required for anti-tumor activity, and the ADC exhibited favorable pharmacokinetics after repeat doses. Together these data support the clinical development of XMT-2056. Citation Format: Jeremy R. Duvall, Raghida A. Bukhalid, Naniye M. Cetinbas, Kalli C. Catcott, Kelly Slocum, Kenneth Avocetien, Keith W. Bentley, Stephen Bradley, Susan Clardy, Scott D. Collins, Elizabeth Ditty, Timothy Eitas, Brian D. Jones, Eugene W. Kelleher, Winnie Lee, Travis Monnell, Rebecca Mosher, Marina Protopopova, LiuLiang Qin, Pamela Shaw, Elena Ter-Ovanesyan, Joshua D. Thomas, Phonphimon Wongthida, Ling Xu, Liping Yang, Jeffrey Zurita, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-2056, a well-tolerated, Immunosynthen-based STING-agonist antibody-drug conjugate which induces anti-tumor immune activity [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 1738.

STING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an innate anti-tumor immune response. While in principle systemic administration of a STING agonist would have many therapeutic benefits, including the delivery of STING to all tumor lesions, such an approach may be limited by toxicity. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that is ideally suited to enable systemic administration without associated toxicity concerns via a targeted delivery strategy. Herein, we demonstrate that systemically administered STING agonist ADCs have greater anti-tumor activity as well as greatly improved tolerability compared to an intravenously (IV) administered, unconjugated (free) agonist. We generated novel STING agonist ADCs by leveraging our Immunosynthen platform, in which the chemical scaffold for bioconjugation is optimized for the STING agonist, resulting in an ADC that has desirable physicochemical and drug-like properties. We have studied the in vitro activity and mechanism of action of STING agonist ADCs in monoculture and co-culture systems. STING agonist ADCs were at least 100-fold more potent in inducing interferon and cytokines as well as tumor cell-killing relative to free agonist. STING agonist ADCs against several targets (antigens) have been evaluated for anti-tumor activity and pharmacodynamic and pharmacokinetic properties in multiple xenograft and syngeneic models. A single administration of STING agonist ADC resulted in target-dependent, durable, and complete regressions. Importantly, the STING agonist ADC led to an increase in tumor-localized inflammatory cytokines and significant immune cell infiltration, while levels of systemic cytokines remained low. In contrast, IV administered free agonist induced up to 100-fold higher levels of systemic cytokines with concomitant body weight loss but only modest tumor growth delay. In summary, Immunosynthen represents a novel STING agonist ADC platform. We have demonstrated target-dependent anti-tumor immune responses in vitro and in vivo for multiple targets, tumor models, and mouse strains. In each case the STING agonist ADC was more active and better tolerated than the IV administered free agonist. Citation Format: Raghida A. Bukhalid, Jeremy R. Duvall, Naniye Malli Cetinbas, Kalli C. Catcott, Kenneth Avocetien, Keith W. Bentley, Stephen Bradley, Tyler Carter, Chen-Ni Chin, Susan Clardy, Scott D. Collins, Timothy Eitas, Brian D. Jones, Eugene W. Kelleher, Rebecca Mosher, Mark Nazzaro, Marina Protopopova, Pamela Shaw, Kelly Slocum, Elena Ter-Ovanesyan, LiuLiang Qin, Joshua D. Thomas, Ling Xu, Liping Yang, Jeffrey Zurita, Dorin Toader, Marc Damelin, Timothy B. Lowinger. Systemic administration of STING agonist antibody-drug conjugates elicit potent anti-tumor immune responses with minimal induction of circulating cytokines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6706.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Targeting the stromal cell-derived factor 1α (SDF-1α)/C-X-C chemokine receptor type 4 (CXCR4) axis has been shown to be a promising therapeutic approach to overcome chemoresistance in acute myeloid leukemia (AML). We investigated the antileukemia efficacy of a novel peptidic CXCR4 antagonist, LY2510924, in preclinical models of AML. LY2510924 rapidly and durably blocked surface CXCR4 and inhibited stromal cell-derived factor 1 (SDF-1)α-induced chemotaxis and prosurvival signals of AML cells at nanomolar concentrations more effectively than the small-molecule CXCR4 antagonist AMD3100. In vitro, LY2510924 chiefly inhibited the proliferation of AML cells with little induction of cell death and reduced protection against chemotherapy by stromal cells. In mice with established AML, LY2510924 caused initial mobilization of leukemic cells into the circulation followed by reduction in total tumor burden. LY2510924 had antileukemia effects as monotherapy as well as in combination with chemotherapy. Gene expression profiling of AML cells isolated from LY2510924-treated mice demonstrated changes consistent with loss of SDF-1α/CXCR4 signaling and suggested reduced proliferation and induction of differentiation, which was proved by showing the attenuation of multiple prosurvival pathways such as PI3K/AKT, MAPK, and β-catenin and myeloid differentiation in vivo. Effective disruption of the SDF-1α/CXCR4 axis by LY2510924 may translate into effective antileukemia therapy in future clinical applications.

Antibody-drug conjugates (ADCs) consist of a tumor-targeted antibody, a drug (payload) with specified mechanism of action, and the chemical framework for attaching them to each other. By selective delivery of the payload to the tumor and not to normal tissues, ADCs can provide greater efficacy and tolerability than systemic chemotherapies, which can translate to longer duration of treatment and response, as well as more options for combination therapies without the concern of overlapping toxicity. To preempt potential resistance to therapy, we have engineered a dual-payload ADC (DP-ADC) that delivers two mechanistically distinct payloads to a single target cell. To build a precision dual payload-ADC (DP-ADC), we leveraged our Synthemer platform, which enables us to chemically attach the payloads to a synthetic scaffold in a defined manner, and then to chemically attach the loaded scaffold to the antibody. This approach allows for unambiguous control of architecture and ratio of the payloads: the payloads are incorporated in a fixed ratio into the scaffold and subsequently into the ADC. Moreover, the synthetic design also allows for selection of solubilizing groups and charge compensation for each payload, allowing for optimal properties of the ADC. Our prototype DP-ADC combines the microtubule inhibitor auristatin F hydroxypropyl amide (AF-HPA) with the DNA monoalkylator I-BiP (related to pyrrolobenzodiazepine), at a payload ratio of 3:1. The drug-to-antibody ratio (DAR) was 12 AF-HPA and 4 I-BiP (16 total), achieved by conjugating 4 of the loaded dual-payload scaffolds to the antibody. The DP-ADC exhibited comparable antigen binding to the unconjugated antibody, and cytotoxic potency comparable to both single-payload ADCs alone. Using cell-based assays that specifically probe the mechanism of action of each payload, we demonstrated that the DP-ADC exerted both expected mechanisms of action on the tumor cell in a target-dependent manner. Considerations for the selection of payload combinations will also be presented. While synergistic action is typically assumed to be preferred if not required, a recent study demonstrated that the observed clinical benefit of most combinations can be explained by the drugs’ independent modes of action, not synergy. Indeed, AF-HPA and I-BiP did not exhibit synergy in cytotoxicity studies in cancer cell lines, yet their combination in a DP-ADC is still expected to confer potential clinical benefit over either single-payload ADC across a patient population. Citation Format: Jeremy R. Duvall, Marc Damelin, Mariya V. Kozytska, Barrett J. Nehilla, Marina Protopopova, Patrick R. Conlon, LiuLiang Qin, Mark Nazzaro, Josh D. Thomas, Qingxiu Zhang, Dorin Toader, Timothy B. Lowinger. An antibody-drug conjugate carrying a microtubule inhibitor and a DNA alkylator exerts both mechanisms of action on tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 232.

A phospholipid-based nanoemulsion formulation of SQ641 (SQ641-NE) was active against intracellular Mycobacterium tuberculosis in J774A.1 mouse macrophages, although SQ641 by itself was not. Intravenous (i.v.) SQ641-NE was cleared from circulation and reached peak concentrations in lung and spleen in 1 h. In a murine tuberculosis (TB) model, 8 i.v. doses of SQ641-NE at 100 mg/kg of body weight over 4 weeks caused a 1.73 log 10 CFU reduction of M. tuberculosis in spleen and were generally bacteriostatic in lungs.

Large-scale genomic profiling efforts, such as The Cancer Genome Atlas (TCGA) have painted an unprecedentedly detailed picture of the genetic alterations that underlie carcinogenesis, yet the key challenge remains as to how to turn this information into actionable therapies. Genomic deletions are a frequent event in diverse cancers, which inactivate a limited number of tumor suppressor genes (“driver”-events) but frequently include many chromosomal neighbors as “passengers”, some of which play critical but redundant roles in normal cellular housekeeping. The overall hypothesis we propose to test is that collateral deletions of such “passenger” genes can be utilized as novel targets of synthetic lethality, an idea which we term “collateral lethality." The large number of passenger deleted genes, playing diverse functions in cell homeostasis, offers a rich repertoire of pharmacologically targetable vulnerabilities presenting novel opportunities for the development of personalized anti-neoplastic therapies. We have provided in vitro proof-of-principal of a collaterally deleted glycolytic gene Enolase 1 (ENO1) at the 1p36 tumor suppressor locus in glioblastoma (GBM), that leads to dramatic sensitization to inhibition of the redundant paralogue, ENO2. The next step is to take this concept to the clinic. Our overall goal is to generate a clinical candidate Enolase inhibitor for tumors with ENO1-deletions, like GBM for which there is no other treatment option and the therapeutic benefit could be quite substantial. Citation Format: Yu-Hsi Lin, Naima Hammoudi, Nikunj Satani, Jeffrey Ackroyd, Sunada Khadka, Dimitra Georgiou, Joe Marszalek, Yuting Sun, Marina Protopopova, Maria E. Di Francesco, Barbara Czako, Alan Y. Wang, Ronald A. DePinho, Florian L. Muller. Collateral lethality: A new target for personalized oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2831.

Antibody-drug conjugates (ADCs) are designed to bind tumor-associated antigens and deliver conjugated cytotoxic payloads to antigen-positive cells. Some ADCs also kill neighboring cells (including antigen-negative cells) by a mechanism referred to as the bystander effect. This effect can be beneficial when the antigen has heterogeneous expression among cells in a solid tumor, but it can also increase off-target toxicity of ADCs. Herein, we report on a unique pharmacologic property of the Dolaflexin platform, which provides a controlled bystander effect that retains the benefits of the bystander effect with respect to antitumor cytotoxicity but reduces the off-target toxicity. The controlled bystander effect, termed “Dolalock,” was achieved through design of a payload, auristatin F-hydroxypropylamide (AF-HPA), that is membrane-permeable and capable of bystander killing but is further catabolized to membrane-impermeable auristatin F (AF). This catabolism of the payload “locks” the highly potent AF in the cell. Using Dolaflexin-based ADCs, we investigated the extent of intracellular AF-HPA and AF release, tumor cell retention and bystander activities in vitro and in vivo. We observed both auristatin species within cells. Co-culture assays with HER2-positive and HER2-negative cells confirmed the cell permeability and bystander-killing capabilities of AF-HPA released from a Dolaflexin-based ADC. Biodistribution studies of Dolaflexin-based ADCs revealed time-dependent concentrations of AF-HPA and AF as well as significant accumulation of AF in xenografted tumor cells, consistent with the Dolalock mechanism. An additional benefit of AF formation was seen in multidrug-resistant transporter studies, which demonstrate that AF, in contrast to AF-HPA, is not a P-glycoprotein 1 (Pgp) substrate. This property may offer additional benefit in Pgp-expressing tumors. In summary, we have shown that the novel AF-HPA payload used in the Dolaflexin platform allows for a controlled bystander effect that likely contributes to the enhanced efficacy and lack of neutropenia we have observed with Dolaflexin-based ADCs. Citation Format: Susan M. Clardy, Alex Yurkovetskiy, Mao Yin, Dmitry Gumerov, Ling Xu, Elena Ter-Ovanesyan, Charlie Bu, Alex Johnson, Marina Protopopova, Qingxiu Zhang, Natalya Bodyak, Marc Damelin, David H. Lee, Donald Bergstrom, Timothy B. Lowinger. Unique pharmacologic properties of Dolaflexin-based ADCs—a controlled bystander effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 754.

Over the past few years we and others reported that specific populations of tumor cells including AML, subsets of lymphoma, glioblastoma, triple negative breast cancer (TNBC), melanoma and pancreatic ductal adenocarcinoma (PDAC) are highly dependent upon oxidative phosphorylation (OXPHOS) to meet their energy and biomass needs. Inhibition of OXPHOS in the context of these dependent tumor populations represents therefore an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign we discovered IACS-010759, a potent, selective small molecule inhibitor of complex I of the mitochondria electron transport chain that possesses excellent pharmacokinetic (PK) and pharmacologic properties, making it suitable for clinical development. We advanced IACS-010759 through IND studies and have recently initiated Phase I studies in patients with relapsed/refractory acute myeloid leukemia (AML) and advanced solid tumors and lymphomas (NCT02882321 and NCT03291938). In this presentation we will describe the identification of a novel series of Complex I inhibitors and their optimization into the clinical candidate compound IACS-010759. Several challenges were successfully overcome, including the optimization of the pharmacokinetic profile and the identification of inhibitors with minimal activity shift across preclinical species, thus enabling a thorough evaluation of the efficacy and toxicology profile. Aspects of the extensive translational research conducted to elucidate the mechanism of action of IACS-010759 and to position it into the clinic will be discussed, including the compelling pharmacological response observed in multiple PDX models of primary AML, and PDX xenograft models of lymphoma, TNBC, glioblastoma, melanoma and PDAC. The observed response was associated with robust pharmacodynamic read-out as assessed by modulation of oxygen consumption rate (OCR), aspartate and specific transcriptional changes. The presentation will also cover the preclinical development activities which resulted in IACS-010759 advancing into on-going phase 1 evaluation in AML and solid tumors. Citation Format: Maria Emilia Di Francesco, Joseph R. Marszalek, Timothy McAfoos, Christopher L. Carroll, Zhijun Kang, Gang Liu, Jay P. Theroff, Jennifer P. Bardenhager, Madhavi L. Bandi, Jennifer R. Molina, Sonal Gera, Marina Protopopova, Yuting Sun, Mary K. Geck Do, Ningping Feng, Jason P. Gay, Florian Muller, Marina Konopleva, Funda Meric-Bernstam, Carlo Toniatti, Timothy P. Heffernan, Giulio F. Draetta, Philip Jones. Discovery and development of IACS-010759, a novel inhibitor of Complex I currently in phase I studies to exploit oxidative phosphorylation dependency in acute myeloid leukemia and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1655.

Antibody-drug conjugates (ADCs) are a highly potent class of drugs that specifically target cancer cells expressing a tumor associated antigen. The ADC TAK-522 consists of a novel human IgG1 anti-HER2 monoclonal antibody and a novel, auristatin-based cytotoxic payload (Auristatin F-hydroxypropylamide, AF-HPA). An average DAR of twelve AF-HPA molecules is achieved via a biodegradable polymer conjugation platform. The free payload AF-HPA and TAK-522 induced immunogenic cell death (ICD) in vitro in multiple cell lines measured by cell surface expression of calreticulin using microscopy and flow cytometry. Furthermore, we developed a novel syngeneic breast cancer model expressing human HER2 at a relatively low antigen density. Treatment in this poorly immunogenic tumor model with TAK-522 but not Kadcyla showed significant inhibition of tumor growth in vivo. Importantly, a combination of anti-PD1 mAb and TAK-522 therapy substantially and synergistically enhanced the anti-tumor efficacy, resulting in complete responses in some mice. The frequency of complete responders was further increased when the two drugs were sequentially, rather than concurrently, administered such that TAK-522 administration was followed by anti-PD1 mAb therapy, suggesting an immunological mechanism with induction of immunogenic cell death by TAK-522, which may activate the adaptive immune system by releasing tumor specific antigens. TAK-522 is currently being tested in a phase-1b clinical trial in patients with advanced breast, lung and gastric cancer expressing HER2. Based on our data, TAK-522 represents a potential candidate for combination therapies with immune checkpoint modulators in patients with poorly immunogenic HER2 expressing tumors.

As part of our ongoing research effort to develop new therapeutics for treatment of tuberculosis (TB), we synthesized a combinatorial library of 10,358 compounds on solid support using a pool-and-split technique and tested the resulting compounds for activity against Mycobacterium tuberculosis. Structure–activity relationship (SAR) evaluation identified new compounds with antitubercular activity, including a novel hit series that is structurally unrelated to any existing antitubercular drugs, dipiperidines. Dipiperidine representatives exhibited MIC values as low as 7.8 μM, the ability to induce promoter Rv0341 activated in response to cell wall biosynthesis inhibition, relatively low nonspecific cellular toxicity in the range of 30–162 μM, and log P values less than 4.

The last 10 years have seen resurgent industry activity in discovery and development of new drugs for the treatment of tuberculosis (TB), a growing widespread and devastating (more than 2 million deaths annually) bacterial infection that is of increasing concern in developing and developed nations alike. This review describes drugs currently being evaluated in the clinic for treatment of uncomplicated and drug resistant pulmonary TB, and updates the literature on 5 new drugs that entered clinical trials in the last 4 years.

OBJECTIVES Clostridium difficile infection (CDI) is a primary cause of antibiotic-associated diarrhoeal illness. Current therapies are insufficient as relapse rates following antibiotic treatment range from 25% for initial treatment to 60% for treatment of recurrence. In this study, we looked at the efficacy of SQ641 in a murine model of CDI. SQ641 is an analogue of capuramycin, a naturally occurring nucleoside-based compound produced by Streptomyces griseus. METHODS In a series of experiments, C57BL/6 mice were treated with a cocktail of antibiotics and inoculated with C. difficile strain VPI10463. Animals were treated orally with SQ641 for 5 days at a dose range of 0.1-300 mg/kg/day, 20 mg/kg/day vancomycin or drug vehicle. Animals were monitored for disease severity, clostridial shedding and faecal toxin levels for 14 days post-infection. RESULTS Five day treatment of CDI with SQ641 resulted in higher 14 day survival rates in mice compared with either vancomycin or vehicle alone. CDI survival rates were 100% (13 of 13) and 94% (32 of 34), respectively, in the 1 and 10 mg/kg/day SQ641 treatment groups, 37% (7 of 19) with vancomycin treatment at 20 mg/kg/day and 32% (14 of 44) in the vehicle-only control group. Secondary measures of efficacy, such as prevention of weight loss, decreased disease severity, decreased C. difficile shedding and decreased toxin in faeces, were observed with SQ641 and vancomycin treatment. CONCLUSIONS SQ641 is effective for CDI treatment with prevention of relapse in the murine model of CDI.

Purpose: To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models. Experimental Design: Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models. Results: Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a “hypoxia index” compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo. In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells. Conclusions: These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins. Clin Cancer Res; 22(7); 1687–98. ©2015 AACR.

Objectives: The purpose of this study was to determine interactions of SQ109, a new asymmetric diamine tuberculosis (TB) drug candidate, with existing antitubercular drugs in vitro and assess its potential to improve combination drug activities against Mycobacterium tuberculosis. Methods: Two-drug combinations at various concentrations below their MICs were tested for growth inhibition of M. tuberculosis using the BACTEC 460 system in vitro. Drug interactions were evaluated based on the quotient values that were derived numerically from the growth indices of cultures treated with a single antibiotic or combination treatment with two antibiotics. Results: SQ109at0.5 of its MIC demonstrated strong Synergistic activity with 0.5 MIC isoniazid and as low as 0.1 MIC rifampicin in inhibition of M. tuberculosis growth. Additive effects were observed between SQ109 and streptomycin, but neither synergy nor additive effects were observed with the combination of SQ109 with ethambutol or pyrazinamide. The synergy between SQ109 and rifampicin was also demonstrated using rifampicin-resistant (RIF R ) M. tuberculosis strains; SQ109 lowered the MIC of rifampicin for these drug-resistant strains. Conclusions: SQ109 interacts Synergistically with isoniazid and rifampicin, two of the most important front-line TB drugs. This finding supports efforts to further evaluate new combination therapies containing SQ109 in experimental animal models of TB that emulate future clinical trial studies in humans.

SummaryHistiocytic sarcoma (HS) is a rare malignant proliferation of histiocytes of uncertain molecular pathogenesis. Here, genetic analysis of coincident loss of Pten and Ink4a/Arf tumor suppressors in the mouse revealed a neoplastic phenotype dominated by a premalignant expansion of biphenotypic myelolymphoid cells followed by the development of HS. Pten protein loss occurred only in the histiocytic portion of tumors, suggesting a stepwise genetic inactivation in the generation of HS. Similarly, human HS showed genetic or epigenetic inactivation of PTEN, p16INK4A, and p14ARF, supporting the relevance of this genetically engineered mouse model of HS. These genetic and translational observations establish a cooperative role of Pten and Ink4a/Arf in the development of HS and provide mechanistic insights into the pathogenesis of human HS.

A diverse 5000-compound library was synthesized from commercially available diamines and screened for activity against Mycobacterium tuberculosis in vitro, revealing 143 hits with minimum inhibitory concentration (MIC) equal to or less than 12.5 microM. New prospective scaffolds with antitubercular activity derived from homo-piperazine, phenyl- and benzyl-substituted piperazines, 4-aminomethylpiperidine, 4-aminophenylethylamine, and 4,4'-methylenebiscyclohexylamine were identified. Compound SQ775 derived from homopiperazine and compound SQ786 derived from benzylpiperazine had potent antimicrobial activity against M. tuberculosis in experimental animals in vivo.

SummaryTo identify genetic events underlying the genesis and progression of multiple myeloma (MM), we conducted a high-resolution analysis of recurrent copy number alterations (CNAs) and expression profiles in a collection of MM cell lines and outcome-annotated clinical specimens. Attesting to the molecular heterogeneity of MM, unsupervised classification using nonnegative matrix factorization (NMF) designed for array comparative genomic hybridization (aCGH) analysis uncovered distinct genomic subtypes. Additionally, we defined 87 discrete minimal common regions (MCRs) within recurrent and highly focal CNAs. Further integration with expression data generated a refined list of MM gene candidates residing within these MCRs, thereby providing a genomic framework for dissection of disease pathogenesis, improved clinical management, and initiation of targeted drug discovery for specific MM patients.

This study aimed at characterizing the interspecies absorption, distribution, metabolism and elimination (ADME) profile of N-geranyl-N'-(2-adamantyl)ethane-1,2-diamine (SQ109), a new diamine-based antitubercular drug. Single doses of SQ109 were administered (intravenously (i.v.) and per os (p.o.)) to rodents and dogs and blood samples were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). Based on i.v. equivalent body surface area dose, the terminal half-life (t1/2) of SQ109 in dogs was longer than that in rodents, reflected by a larger volume of distribution (Vss) and a higher clearance rate of SQ109 in dogs, compared to that in rodents. The oral bioavailability of SQ109 in dogs, rats and mice were 2.4-5, 12 and 3.8%, respectively. After oral administration of [14C]SQ109 to rats, the highest level of radioactivity was in the liver, followed by the lung, spleen and kidney. Tissue-to-blood ratios of [14C]SQ109 were greater than 1. Fecal elimination of [14C]SQ109 accounted for 22.2% of the total dose of [14C]SQ109, while urinary excretion accounted for only 5.6%. The binding of [14C]SQ109 (0.1-2.5 microg ml-1) to plasma proteins varied from 6 to 23% depending on the species (human, mouse, rat and dog). SQ109 was metabolized by rat, mouse, dog and human liver microsomes, resulting in 22.8, 48.4, 50.8 or 58.3%, respectively, of SQ109 remaining after a 10-min incubation at 37 degrees C. The predominant metabolites in the human liver microsomes gave intense ion signals at 195, 347 and 363m/z, suggesting the oxidation, epoxidation and N-dealkylation of SQ109. P450 reaction phenotyping using recombinant cDNA-expressed human CYPs in conjunction with specific CYP inhibitors indicated that CYP2D6 and CYP2C19 were the predominant CYPs involved in SQ109 metabolism.

Integrating combinatorial lead optimization of [1,2]-diamine core structure based on ethambutol with high-throughput screening has led us to focus on three promising analogs (SQ37, SQ59 and SQ109) as potential anti-tubercular drug candidates from thousands of synthesized diamine analogs for further characterization of their biopharmaceutical and pharmacokinetic properties by using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and cassette dosing for pharmacokinetic screening. Simultaneous separation of the three analogs was achieved on reversed phase HPLC using a gradient mobile phase composed of MeOH/CH(3)COONH(4) (5mM)/trifluoroacetic acid: 80/20/0.1 (v/v/v). After extraction with acetonitrile from biomatrices, samples were analyzed on the LC/MS/MS system in the positive mode using an electrospray ion source. The retention time for the analogs ranged from 3.70 to 4.48 min. Incubation of SQ37 with plasma at 37 degrees C for 6h resulted in its degradation in human and rat plasma (20-35%), but no significant degradation was observed in mouse and dog plasma. SQ59 was relatively stable in the plasma of the four species. SQ109 was degraded in human and dog plasma (30-40%), but stable in mouse and rat plasma during the 6h incubation. A rapid multiple pharmacokinetic screening was taken by cassette dosing of the three analogs to mice and simultaneous analysis of their plasma concentrations. The analogs showed large Vd(ss) ranging from 11,300 (SQ37), 12,800 (SQ109) to 63,900 ml/kg (SQ59). The clearance ranged from 3240 (SQ109), 3530 (SQ37) and 8043 ml/kg/h (SQ59). The elimination t(1/2) ranged from 4.4 to 21.1h dependent on the routes. The oral bioavailability was 5.1 (SQ59), 20.1 (SQ37) and 7.8% (SQ109), respectively. Both SQ37 and SQ109 possess good pharmacokinetic properties.

We used exposure to microwaves from a global system for mobile communication (GSM) mobile phone (915 MHz, specific absorption rate (SAR) 37 mW/kg) and power frequency magnetic field (50 Hz, 15 muT peak value) to investigate the response of lymphocytes from healthy subjects and from persons reporting hypersensitivity to electromagnetic field (EMF). The hypersensitive and healthy donors were matched by gender and age and the data were analyzed blind to treatment condition. The changes in chromatin conformation were measured with the method of anomalous viscosity time dependencies (AVTD). 53BP1 protein, which has been shown to colocalize in foci with DNA double strand breaks (DSBs), was analyzed by immunostaining in situ. Exposure at room temperature to either 915 MHz or 50 Hz resulted in significant condensation of chromatin, shown as AVTD changes, which was similar to the effect of heat shock at 41 degrees C. No significant differences in responses between normal and hypersensitive subjects were detected. Neither 915 MHz nor 50 Hz exposure induced 53BP1 foci. On the contrary, a distinct decrease in background level of 53BP1 signaling was observed upon these exposures as well as after heat shock treatments. This decrease correlated with the AVTD data and may indicate decrease in accessibility of 53BP1 to antibodies because of stress-induced chromatin condensation. Apoptosis was determined by morphological changes and by apoptotic fragmentation of DNA as analyzed by pulsed-field gel electrophoresis (PFGE). No apoptosis was induced by exposure to 50 Hz and 915 MHz microwaves. In conclusion, 50 Hz magnetic field and 915 MHz microwaves under specified conditions of exposure induced comparable responses in lymphocytes from healthy and hypersensitive donors that were similar but not identical to stress response induced by heat shock.

SQ109 is a novel [1,2]-diamine-based ethambutol (EMB) analog developed from high-throughput combinatorial screening. The present study aimed at characterizing its pharmacodynamics and pharmacokinetics. The antimicrobial activity of SQ109 was confirmed in vitro (Mycobacterium tuberculosis-infected murine macrophages) and in vivo (M. tuberculosis-infected C57BL/6 mice) and compared to isoniazid (INH) and EMB. SQ109 showed potency and efficacy in inhibiting intracellular M. tuberculosis that was similar to INH, but superior to EMB. In vivo oral administration of SQ109 (0.1-25 mg kg(-1) day(-1)) to the mice for 28 days resulted in dose-dependent reductions of mycobacterial load in both spleen and lung comparable to that of EMB administered at 100 mg kg(-1) day(-1), but was less potent than INH at 25 mg kg(-1) day(-1). Monitoring of SQ109 levels in mouse tissues on days 1, 14 and 28 following 28-day oral administration (10 mg kg(-1) day(-1)) revealed that lungs and spleen contained the highest concentration of SQ109, at least 10 times above its MIC. Pharmacokinetic profiles of SQ109 in mice following a single administration showed its C(max) as 1038 (intravenous (i.v.)) and 135 ng ml(-1) (p.o.), with an oral T(max) of 0.31 h. The elimination t(1/2) of SQ109 was 3.5 (i.v.) and 5.2 h (p.o.). The oral bioavailability was 4%. However, SQ109 displayed a large volume of distribution into various tissues. The highest concentration of SQ109 was present in lung (>MIC), which was at least 120-fold (p.o.) and 180-fold (i.v.) higher than that in plasma. The next ranked tissues were spleen and kidney. SQ109 levels in most tissues after a single administration were significantly higher than that in blood. High tissue concentrations of SQ109 persisted for the observation period (10 h). This study demonstrated that SQ109 displays promising in vitro and in vivo antitubercular activity with favorable targeted tissue distribution properties.

In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53-HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.

Epstein-Barr virus (EBV) carrying lymphoblastoid cells of normal origin express the full program of all 9 virus-encoded, growth transformation associated proteins. They have an intact p53 pathway as a rule. This raises the question of whether any of the viral proteins impair the pathway functionally. Using a yeast 2-hybrid system, we have shown that EBNA-5 but not the other EBNAs interacts with the p14ARF protein, a regulator of the p53 pathway. The interaction was confirmed in vitro using a GST pull-down assay. Moreover, expression of EBNA-5 increased the survival of p14ARF-transfected cells. EBV infection of resting B cells induced the expression of p14ARF mRNA without increased level of the protein. A fraction of the p14ARF localized to the nucleoli but the bulk of the protein accumulated in nuclear but extranucleolar inclusions. Formation of the extranucleolar inclusions led to complete relocalization of EBNA-5 from nucleoplasm to these structures. The inclusions also contained p53 and HDM2, and were surrounded by PML bodies and proteasomes, which suggests that these inclusions could be targets for proteasome dependent protein degradation. © 2003 Wiley-Liss, Inc.

Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production to support cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-010759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that AML, plus subsets of glioblastoma, neuroblastoma, lymphoma, melanoma, triple negative breast cancer (TNBC) and pancreatic cancer (PDAC) are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in several cancer types with IACS-010759 led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. Mechanistically, response to IACS-010759 was associated with induction of a metabolic imbalances that negatively impacted energy homeostasis, aspartate biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. Tumor growth inhibition and regression have been observed in molecularly defined subsets of TNBC and PDAC PDX xenograft models treated with IACS-010759, indicating that subsets of these indications are dependent on OXPHOS. Furthermore, treating TNBC or PDAC PDX models post-chemotherapy with IACS-010759 extends progression free survival, consistent with IACS-010759 targeting recently described metabolically adapted residual tumor cells. In orthotopic xenograft models of primary AML cells, daily oral treatment with 1-7.5 mg/kg IACS-010759 extended the median survival. Efficacy was paralleled by robust modulation of OCR, aspartate, and a gene signature levels. Therefore, these readouts (OCR, aspartate and a nanostring geneset) have been validated for use as exploratory clinical biology of response endpoints. In parallel, completion of preclinical chemistry, manufacturing and control (CMC) as well as GLP safety and tolerability studies with IACS-010759 in multiple species have enabled the selection of a clinical entry dose. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, a Phase I clinical trial in relapsed, refractory AML was initiated in October 2016, with a parallel trial in solid tumors expected to initiate in early 2017. Initial results from the on-going AML trial will be disclosed. Citation Format: Jennifer Molina, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Jason Cross, Naval Daver, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Jing Han, Judy Hirst, Sha Huang, Yongying Jiang, Zhijun Kang, Marina Konopleva, Gang Liu, Helen Ma, Polina Matre, Timothy McAfoos, Funda Meric-Bernstam, Pietro Morlacchi, Florian Muller, Marina Protopopova, Melinda Smith, Sonal Sonal, Yuting Sun, Jay Theroff, Andrea Viale, Quanyun Xu, Carlo Toniatti, Giulio Draetta, Philip Jones, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759, a novel inhibitor of complex I in Phase I clinical development to target OXPHOS dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4971. doi:10.1158/1538-7445.AM2017-4971

Tumor cells normally depend on both glycolysis and oxidative phosphorylation (OXPHOS) to provide the energy and macromolecule building blocks needed to enable continued tumor cell growth. Genetic or epigenetic inactivation of one of these two redundant pathways represents a metabolic vulnerability that should be susceptible to an inhibitor of the other pathway. We have identified multiple contexts where all or a subset of these tumors demonstrate a dependence on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent inhibitor of complex I of oxidative phosphorylation. In isolated mitochondria or permeabilized cells, ATP production or oxygen consumption is inhibited at single digit nM concentrations in the presence of malate/glutamate, but not succinate. More directly, IACS-10759 inhibits the conversion of NADH to NAD+ in an immunoprecipitated complex I assay at low nM concentrations. Importantly, IACS-10759 is orally bioavailable with excellent pharmacokinetics properties in preclinical species, and has an overall profile suitable for clinical development. Our group and others have demonstrated that a variety of tumor types including: AML, plus subsets of lymphoma, breast, melanoma and PDAC are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in multiple cancer types with IACS-10759 led to decreased oxygen consumption rate (OCR). IACS-10759 treatment also led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. In multiple PDX models of primary AML IACS-10759 treatment extends the median survival. Efficacy was paralleled by robust modulation of OCR, aspartate, and p-AMPK levels. Additionally, tumor growth inhibition or regression was also observed in cell line and PDX xenograft models of lymphoma, triple negative breast, melanoma and PDAC treated with IACS-10759, indicating that subsets of several non-AML indications are also dependent on OXPHOS. Mechanistically, extensive metabolic profiling revealed that the response to IACS-10759 was associated with induction of a metabolic imbalances that negatively impacted energy homeostasis, amino acid biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. As a result of the robust preclinical response in multiple model systems, IACS-10759 has been advanced through IND enabling studies. GLP safety and toxicology have been completed, clinical supplies manufactured, and a Phase I clinical trial in AML will be initiated during the second quarter of 2016. This abstract is also being presented as Poster B35. Citation Format: Philip Jones, M Emilia Di Francesco, Jennifer M. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher A. Bristow, Christopher L. Carroll, Ningping Feng, Jason P. Gay, Mary K. Geck Do, Jennifer M. Greer, Marina Konopleva, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda G. Smith, Sonal Fnu, Jay P. Theroff, Giulio Draetta, Giulio Draetta, Carlo Toniatti, Joseph R. Marszalek. IACS-010759 a novel inhibitor of oxidative phosphorylation advancing into first-in-human studies to exploit metabolic vulnerabilities. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr PR01.

Substitution of the new diamine antibiotic SQ109 for ethambutol in a mouse model of chronic tuberculosis (TB) improved efficacy of combination drug therapy with first-line TB drugs rifampin and isoniazid, with or without pyrazinamide: at 8 weeks, lung bacteria were 1.5 log 10 lower in SQ109-containing regimens.

Large scale genomic characterization efforts such as TCGA have painted an unprecedentedly detailed picture of the genetic alterations that underlie tumorigenesis. Yet, the majority of genetic alterations are passenger rather than driver events and are considered unactionable. We have previously proposed that passenger or collateral deletions could serve as pharmacologically targetable vulnerabilities Collateral Lethality, in case passenger genes are homozygously deleted and member of a paralogous gene family carrying out an essential housekeeping function. We have presented proof-of-principal, whereby passenger deletion of the glycolytic gene Enolase 1 (ENO1) as part of the 1p36-tumor suppressor locus, renders glioma cells harboring such deletions highly sensitive to ablation of its redundant paralogue, ENO2. While our original analysis identified ENO1-homozygous deletions in Glioblastoma (GBM), recent bioinformatics analyzes, backed by immunohistochemistry, show that ENO1-homozygous deletions also occur in Hepatocellular Carcinoma and Cholangiocarcinoma. In GBM, multisector analysis of primary and recurrent tumors, indicate that ENO1 deletion is an early event which is homogenously distributed through the primary tumor and persist during recurrence. To pharmacologically exploit ENO1-deletion, we have pursued two approaches. First, we have synthesized cell-permeable prodrug derivatives of the natural Enolase inhibitor SF2312. The lead compound, POMHEX, shows potent killing of ENO1-deleted glioma cells in the low nM range while ENO1-restored isogenic or normal cells can tolerate μM doses. POMHEX has a short half-life yet can eradicate intracranial xenografted ENO1-deleted tumors, provided extensive breakdown of the blood-brain barrier. Our second approach to targeting ENO1-deletion consisted of chemical biology screening of drug libraries for the ability to kill ENO1-deleted but not isogenic rescued cells. We find that ENO1-deleted cells show a dramatic sensitization to inhibitors of the mitochondrial electron transport chain. These include tool compounds such as rotenone as well as compounds not previously associated with mitochondria, such as Mubritinib and an experimental anti-neoplastic agent previously described as a HIF1-inhibitor, now known to inhibit mitochondrial Complex I. The latter agent shows potent activity against ENO1-deleted intracranial xenografts. The likely cause for this sensitivity is the inability of ENO1-deleted cells to compensatory upregulate glycolysis in response mitochondrial inhibition, the typical response of ENO1-intact glioma cells and normal cells. Together, these data indicate that passenger deletion of ENO1 is an encouraging drug-target and provide support for collateral lethality as a viable therapeutic strategy, which, given the large number of passenger deletions in the cancer genome, may be broadly applicable. Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Joe Marszalek, Yuting Sun, Marina Protopopova, Maria E. Di Francesco, Barbara Czako, Alan Wang, Ronald A. DePinho, Florian L. Muller. Passenger deletion of ENO1 as a collateral lethality target in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3837.

Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production leading to robust cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that a variety of tumor types including: AML, plus subsets of lymphoma, breast, melanoma and PDAC are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in multiple cancer types with IACS-10759 led to decreased oxygen consumption rate (OCR). IACS-10759 treatment also led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. In an orthotopic xenograft model of primary AML cells derived from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 treatment with 3 and 10 mg/kg orally using a 5 on/2 off schedule extended the median survival by greater than 2-fold. Efficacy was paralleled by robust modulation of OCR, aspartate, and p-AMPK levels. Additionally, tumor growth inhibition or regression was also observed in cell line and PDX xenograft models of lymphoma, triple negative breast, melanoma and PDAC treated with IACS-10759, indicating that subsets of several non-AML indications are also dependent on OXPHOS. Mechanistically, extensive metabolic profiling and flux analysis revealed that the response to IACS-10759 was associated with induction of a metabolic imbalance that negatively impacted energy homeostasis, amino acid biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, IACS-10759 has been advanced through IND enabling studies. GLP safety and toxicology have been completed, and we expect to file an IND at the end of 1Q2016 and initiate a Phase I clinical trial in AML during the second quarter of 2016. Citation Format: Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Jing Han, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Gera, Jay Theroff, Quanyun Xu, Juliana Velez, Carlo Toniatti, Timothy Heffernan, Giulio Draetta, M. Emilia Di Francesco, Philip Jones, Joseph R. Marszalek. Title: IACS-010759 is a novel clinical candidate that targets AML cells by inducing a metabolic catastrophe through inhibition of oxidative phosphorylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 335.

Tumor cells normally depend on both glycolysis and oxidative phosphorylation (OXPHOS) to provide the energy and macromolecule building blocks for rapid growth. Metabolic vulnerabilities caused by inactivation of glycolysis render tumor cells highly dependent on OXPHOS, and represent a therapeutic opportunity. Through an extensive medicinal chemistry campaign, we have identified IACS-10759 as a potent inhibitor of complex I of OXPHOS. IACS-10759 effectively inhibits ATP production and oxygen consumption in isolated mitochondria, and inhibits the conversion of NADH to NAD+ in immunoprecipitated complex I in low nM range. The exact subunit that IACS-10759 binds to is under investigation. Importantly, IACS-10759 is orally bioavailable with excellent physicochemical properties in preclinical species, and shows significant efficacy in multiple tumor indications both in vitro and in vivo. Specifically, in a glycolysis-deficient xenograft model, IACS-10759 causes robust tumor regression, but has no effect in the same model when glycolysis is restored. In addition, in AML where tumor cells have been shown to be highly OXPHOS-dependent, IACS-10759 robustly suppresses cell growth and induces apoptosis in both primary AML samples and cell lines in vitro, but not in normal patient-derived bone marrow cells. Significantly, IACS-10759 extends median survival by over 50 days in an AML orthotopic xenograft model. Furthermore, IACS-10759 also shows selective efficacy in other cell line panels including pancreatic cancer, non-small cell lung cancer and colorectal cancer, and has synergism with glycolysis inhibitors. In light of these results, we are currently performing IND enabling studies for IACS-10759, with first-in-human studies targeted for fourth quarter of 2015. Citation Format: Marina Protopopova, Madhavi Bandi, Yuting Sun, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Marina Konopleva, Polina Matre, Zhijun Kang, Gang Liu, Florian Muller, Timothy Lofton, Timothy McAfoos, Melinda Smith, Jay Theroff, Jing Han, Yuanqing Wu, Lynda Chin, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-10759: A novel OXPHOS inhibitor that selectively kills tumors with metabolic vulnerabilities. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A65.

Existing drugs have limited efficacy against the rising threat of drug-resistant TB, have significant side effects, and must be given in combinations of four to six drugs for at least 6 months for drug-sensitive TB and up to 24 months for drug-resistant TB. The long treatment duration has led to increased patient noncompliance with therapy. This, in turn, drives the development of additional drug resistance in a spiral that has resulted in some forms of TB being currently untreatable by existing drugs. New antitubercular drugs in development, particularly those with mechanisms of action that are different from existing first- and second-line TB drugs, are anticipated to be effective against both drug-sensitive and drug-resistant TB. SQ109 is a new TB drug candidate with a novel mechanism of action that was safe and well tolerated in Phase I and early Phase II clinical trials. We describe herein the identification, development and characterization of SQ109 as a promising new antitubercular drug.

We recently reported that compounds created around a dipiperidine scaffold demonstrated activity against Mycobacterium tuberculosis (Mtb) (Bogatcheva, E.; Hanrahan, C.; Chen, P.; Gearhart, J.; Sacksteder, K.; Einck, L.; Nacy, C.; Protopopova, M. Bioorg. Med. Chem. Lett. 2010, 20, 201). To optimize the dipiperidine compound series and to select a lead compound to advance into preclinical studies, we evaluated the structure–activity relationship (SAR) of our proprietary libraries. The (piperidin-4-ylmethyl)piperidine scaffold was an essential structural element required for antibacterial activity. Based on SAR, we synthesized a focused library of 313 new dipiperidines to delineate additional structural features responsible for antitubercular activity. Thirty new active compounds with MIC 10–20 μg/ml on Mtb were identified, but none was better than the original hits of this series, SQ609, SQ614, and SQ615. In Mtb-infected macrophages in vitro, SQ609 and SQ614 inhibited more than 90% of intracellular bacterial growth at 4 μg/ml; SQ615 was toxic to these cells. In mice infected with Mtb, weight loss was completely prevented by SQ609, but not SQ614, and SQ609 had a prolonged therapeutic effect, extended by 10–15 days, after cessation of therapy. Based on in vitro and in vivo antitubercular activity, SQ609 was identified as the best-in-class dipiperidine compound in the series.

Objectives: To extend capuramycin spectrum of activity beyond mycobacteria and improve intracellular drug activity. Methods: Three capuramycin analogues (SQ997, SQ922 and SQ641) were conjugated with different natural and unnatural amino acids or decanoic acid (DEC) through an ester bond at one or more available hydroxyl groups. In vitro activity of the modified compounds was determined against Mycobacterium spp. and representative Gram-positive and Gram-negative bacteria. Intracellular activity was evaluated in J774A.1 mouse macrophages infected with Mycobacterium tuberculosis (H37Rv). Results: Acylation of SQ997 and SQ641 with amino undecanoic acid (AUA) improved in vitro activity against most of the bacteria tested. Conjugation of SQ922 with DEC, but not AUA, improved its activity against Gram-positive bacteria. In the presence of efflux pump inhibitor phenylalanine arginine β-naphthyl amide, MICs of SQ997-AUA, SQ641-AUA and SQ922-DEC compounds improved even further against drug-susceptible and drug-resistant Staphylococcus aureus. In Gram-negative bacteria, EDTA-mediated permeabilization caused 4- to 16-fold enhancement of the activity of AUA-conjugated SQ997, SQ922 and SQ641. Conjugation of all three capuramycin analogues with AUA improved intracellular killing of H37Rv in murine macrophages. Conclusions: Conjugation of capuramycin analogues with AUA or DEC enhanced in vitro activity, extended the spectrum of activity in Gram-positive bacteria and increased intracellular activity against H37Rv.