Ethika Tyagi

IL-2 is a cytokine clinically approved for the treatment of melanoma and renal cell carcinoma. Unfortunately, its clinical utility is hindered by serious side effects driven by the systemic activity of the cytokine. Here, we describe the design and characterization of a conditionally activated IL-2 prodrug, WTX-124, that takes advantage of the dysregulated protease milieu of tumors. WTX-124 was engineered as a single molecule containing an inactivation domain and a half-life extension domain that are tethered to a fully active IL-2 by protease-cleavable linkers. We show that the inactivation domain prevented IL-2 from binding to its receptors in nontumor tissues, thereby minimizing the toxicity associated with systemic exposure to IL-2. The half-life extension element improves the pharmacokinetic profile of WTX-124 over free IL-2, allowing for greater exposure. WTX-124 was preferentially activated in tumor tissue by tumor-associated proteases, releasing active IL-2 in the tumor microenvironment. In vitro assays confirmed that the activity of WTX-124 was dependent on proteolytic activation, and in vivo WTX-124 treatment resulted in complete rejection of established tumors in a cleavage-dependent manner. Mechanistically, WTX-124 treatment triggered the activation of T cells and natural killer (NK) cells, and markedly shifted the immune activation profile of the tumor microenvironment, resulting in significant inhibition of tumor growth in syngeneic tumor models. Collectively, these data demonstrate that WTX-124 minimizes the toxicity of IL-2 treatment in the periphery while retaining the full pharmacology of IL-2 in the tumor microenvironment, supporting its further development as a cancer immunotherapy treatment.See related Spotlight by Silva, p. 544.

Myelodysplastic syndrome (MDS) is a heterogeneous group of hematopoietic stem cell disorders. Approximately 70% of patients diagnosed with higher-risk (HR) MDS progress to acute myeloid leukemia (AML). Cyclin-dependent kinase 9 (CDK9) influences transcription through phosphorylation and activation of RNA polymerase II (RPB1), which increases the levels of key oncogenic survival genes like myeloid cell leukemia-1 (MCL-1). MCL-1 dependent malignancies can be identified through a functional assay by adding a NOXA mimetic MCL-1 antagonist peptide to patient samples and measuring the subsequent induction of apoptosis. The assay has been tested in bone marrow mononuclear cells (BMMCs) from AML and MDS patients to examine MCL-1 dependency prior to therapy with alvocidib. Approximately 25% of AML patient BMMCs and 60% of MDS patient BMMCs are MCL-1 dependent (data not shown). We hypothesized that the hypomethylating agents (HMAs) azacitidine and decitabine, which are currently approved for ...

BackgroundInterferon α (IFNα) was the first cytokine clinically tested as a cancer therapy. IFNα is a member of the type-I IFN family and activates immune responses either directly by engaging IFNα receptors (IFNAR) ubiquitously expressed on immune cells or indirectly by inducing chemokines that attract myeloid and lymphoid cells to the tumor site. High dose IFNα therapy was approved for melanoma, lymphoma, and leukemia but its use is limited by systemic toxicity and modest efficacy.MethodsWTX-613 is a novel systemically delivered IFNα2b pro-drug identified using the Predator™ discovery platform. The inducible WTX-613 INDUKINE™ molecule is designed to deliver wild-type IFNα2b in the tumor microenvironment to reduce systemic toxicity. WTX-613 has two identical half-life extension (HLE) domains tethered to IFNα2b via a tumor protease-sensitive linker. The HLE domain supports less frequent systemic administration but importantly also prevents binding of WTX-613 to IFNAR due to steric h...

Sumitomo Dainippon Pharma Oncology (f/k/a Tolero Pharmaceuticals), Lehi, UTTNK1 is a poorly understood member of the ACK family of non-receptor tyrosine kinases with a unusual domain arrangement, but no known mechanism of regulation nor conclusive link to disease. We initially identified TNK1 as a mediator of cell survival in a subset of primary patient cancer samples. In an effort to understand how TNK1 is regulated, we discovered a MARK-mediated phosphorylation at S502 near the TNK1 C-terminus that mediates an interaction with 14-3-3. We found that 14-3-3 binding inhibitsTNK1 kinase activity, whereas disruption of this interaction renders TNK1 highly active and capable of driving tumor growth in vivo. In support of this idea, kinase substrate profiling at the proteome level revealed a variety of pro-growth and motility substrates for active TNK1. One unique feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus, which sits adjacent to the 14-3-3 binding site. We...

Interleukin-2 (IL-2) is a cytokine clinically approved for the treatment of melanoma and renal cell carcinoma. Unfortunately, its clinical utility is hindered by serious side effects driven by the systemic activity of the cytokine. Here, we describe the design and characterization of a conditionally activated IL-2 prodrug, WTX-124, that takes advantage of the dysregulated protease milieu of tumors. WTX-124 was engineered as a single molecule containing an inactivation domain and a half-life extension domain that are tethered to a fully active IL-2, by protease-cleavable linkers. We show that the inactivation domain prevented IL-2 from binding to its receptors in non-tumor tissues, thereby minimizing the toxicity associated with systemic exposure to IL-2. The half-life extension element improves the pharmacokinetic profile of WTX-124 over free IL-2, allowing for greater exposure. WTX-124 was preferentially activated in tumor tissue by tumor-associated proteases, releasing active IL-2...

<jats:title>Abstract</jats:title> <jats:p>Systemic therapy with proinflammatory immune modulators to activate anti-tumor immunity is a promising approach to treat cancer. However, poor pharmacokinetic properties and dose-limiting toxicities such as inflammation, cytokine release syndrome, and tissue damage have prevented or limited the clinical use of cytokines such as interleukin 12 (IL-12) and interferon α (IFNα). Previous clinical development of rhIL-12 was terminated due to toxicity, and although rIFNα is approved for the treatment of melanoma, lymphoma, and leukemia, its use has been limited by systemic toxicity and modest efficacy.</jats:p> <jats:p>INDUKINE™ molecules, engineered using Werewolf Therapeutics' Predator™ discovery platform, are novel, systemically delivered cytokine pro-drugs which prevent systemic toxicity and deliver cytokines to the tumor microenvironment (TME) where they are activated. Several INDUKINE™ molecules using Werewolf's proprietary linkers have been previously shown to be cleaved by a wide range of human solid tumors but have yet to be tested in primary human lymphoma samples or syngeneic mouse lymphoma models.</jats:p> <jats:p>IL-12 is a potent, pleiotropic cytokine for immune-mediated killing of cancer cells, whose mechanism of action (MOA) includes activation of both cytotoxic T and NK cells. The WTX-330 INDUKINE™ molecule, a wild-type IL-12 pro-drug, contains a half-life extension (HLE) domain to support infrequent dosing and a high affinity anti-IL-12 neutralizing antibody domain to maintain the molecule in its inactive state in the periphery. Both the HLE and blocking domains are tethered to IL-12 via two identical tumor protease-sensitive linkers. Linker cleavage in the TME removes the HLE domain and the blocker, resulting in active IL-12 within the TME with wild-type IL-12 biologic and PK profiles.</jats:p> <jats:p>IFNα is a member of the type-I IFN family and activates innate immune responses either directly by engaging IFNα receptors (IFNAR) ubiquitously expressed on immune cells or indirectly by inducing chemokines that attract myeloid and lymphoid cells to the tumor site. The WTX-613 INDUKINE™ molecule is an inactive IFNα2b pro-drug with two identical HLE domains tethered to IFNα2b via two identical tumor protease-sensitive linkers. The HLE domains sterically block binding of WTX-613 to IFNAR until cleavage of the linkers in the TME releases active IFNα.</jats:p> <jats:p>Since human IL-12 and IFNα2b are not cross reactive in mice, surrogate WTX-330 and WTX-613 INDUKINE™ molecules were created, consisting of a mouse/human chimeric IL-12 or a mouse IFNα1 to explore anti-tumor responses in syngeneic hematologic cancer models. In the subcutaneously (s.c.) A20 B cell lymphoma model, the WTX-330 surrogate showed dose-dependent anti-tumor activity with 4 out of 10 tumor-free mice at the top dose. The WTX-613 surrogate demonstrated tumor stasis lasting beyond the treatment phase. Utilizing the s.c. EG7.OVA T lymphoblast line, tumor growth was efficiently blocked by the WTX-613 surrogate while the WTX-330 surrogate inhibited tumor growth during the dosing period. Both treatments were well tolerated by the mice at active dose levels.</jats:p> <jats:p>The WTX-330 and WTX-613 surrogates strongly activated NK and CD8+ cell responses and induced APC and effector cell markers in the MC38 syngeneic tumor model supporting a MOA as described for wild-type IL-12 and IFNα. Similar studies are on-going in lymphoma models. PK analysis in mice revealed extended half-life (T1/2) for both WTX-330 and WTX-613 surrogates compared to the short T1/2 of native IL-12 or IFNα1. Finally, WTX-330 and WTX-613 were well tolerated in non-human primates (NHP), resulting in plasma exposure levels for INDUKINE™ molecules that exceeded those needed for anti-tumor activity in mice. In addition, plasma levels of free IL-12 after dosing with WTX-330 were very low compared to tolerated levels of wild-type IL-12. Similar studies for WTX-613 are on-going.</jats:p> <jats:p>Preclinical data obtained so far for both programs support the continued development and future evaluation of these innovative and differentiated therapies in hematologic malignancies, both as monotherapies and in multiple combinations with standard of care.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Isaacs: Werewolf Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Seidel-Dugan: Werewolf Therapeutics: Current Employment, Other: current shareholder .</jats:p> </jats:sec>

Pyruvate kinase functions as the key enzyme in the final step of glycolysis. Cancer cells largely utilize the M2 isoform of pyruvate kinase (PKM2) due to the ability of PKM2 to be allosterically regulated between fully active (tetramer) and less active (dimer) forms of the enzyme. This dynamic regulation is associated with metabolic reprogramming of cancer cells creating a balance between energy needs and anabolic cellular requirements to support cell growth and division. Furthermore, the allosteric regulation creates an opportunity to design a small molecule activator to reverse the metabolic reprogramming favoring cancer growth and immune evasion. TP-1454 is a novel PKM2 activator with low nanomolar PKM2 activation in biochemical assays (AC50 = 10 nM) and in A549 epithelial lung carcinoma cells (AC50 Citation Format: Satya Pathi, Peter Peterson, Ryan Mangelson, Ethika Tyagi, Jason M. Foulks, Clifford J. Whatcott, David J. Bearss, Steven L. Warner. PKM2 activation modulates metabolism and enhances immune response in solid tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B080. doi:10.1158/1535-7163.TARG-19-B080

Prostate cancer is the most prevalent cancer in men, comprising 20% of all new cancer cases in United States, as per the Cancer Statistics, 2019. Many therapies for prostate cancer function by lowering androgen levels and include androgen deprivation therapy alone or in combination with surgical or chemical castration. Hormone therapy has been a mainstay treatment for prostate cancer, ultimately leading to progression free disease in over 80% of patients over short time periods. Unfortunately, these effects are not durable, and the majority of patients experience progressive disease. Ultimately, the disease progresses and becomes invasive and lethal in the form of castration-resistant prostate cancer (CRPC). Understanding the mechanism by which prostate cancer cells lose their inherent dependence on the canonical androgen signaling pathway for survival has been critical in developing new therapeutic options for patients with CRPC. CDK9 may constitute one such mechanism. CDK9 phospho...

Background With 31,000 new cases expected in 2018 (US), and a 50% five-year overall survival rate, there is yet a significant unmet need in the treatment of patients with multiple myeloma (MM). The proteasomal inhibitor bortezomib is approved for the treatment of patients with multiple myeloma. Bortezomib inhibits the degradation of many proteins, including the pro-apoptotic protein NOXA. However, low basal levels of NOXA and/or high levels of the anti-apoptotic protein MCL-1 have been implicated in bortezomib resistance and negative patient outcomes. NOXA functions to sequester MCL-1 and prevent its interaction with the apoptosis inducing proteins, BAK or BAX. The BCL-2 inhibitor, venetoclax, has also been investigated in clinical trials for the treatment of multiple myeloma. Increased MCL-1 expression has been shown to be key in the resistance to venetoclax. Considering the central role of MCL-1 to survival and treatment efficacy in myeloma, we investigated the ability of an MCL-1...

There are conflicting epidemiologic data on whether chronic aspirin (ASA) use may reduce melanoma risk in humans. Potential anti-cancer effects of ASA may be mediated by its ability to suppress prostaglandin E2 (PGE2) production and activate 5'-adenosine monophosphate-activated protein kinase (AMPK). We investigated the inhibitory effects of ASA in a panel of melanoma and transformed melanocyte cell lines, and on tumor growth in a preclinical model. ASA and the COX-2 inhibitor Celecoxib did not affect melanoma cell viability, but significantly reduced colony formation, cell motility, and pigmentation (melanin production) in vitro at concentrations of 1 mM and 20 uM, respectively. ASA-mediated inhibition of cell migration and pigmentation was rescued by exogenous PGE2 or Compound C, which inhibits AMPK activation. Levels of tyrosinase, MITF, and p-ERK were unaffected by ASA exposure. Following a single oral dose of 0.4 mg ASA to NOD-SCID mice, salicylate was detected in plasma an...

Proviral integration site for Moloney murine leukemia virus-1 (Pim-1) is a serine/threonine kinase downstream of Jak/Stat signaling which promotes cell growth, survival, and drug resistance. Pim-1 kinase is an important driver of tumorigenesis and tumor survival through its role in a number of downstream pathways, including inhibition of apoptosis through phosphorylation of the BH3-only protein BAD. Pim-1 is expressed at very low levels in most normal tissues, but is overexpressed in many cancers, such as prostate, colorectal, and many hematologic malignancies. Pim-1 kinase activity is constitutive and therefore directly proportional to protein expression. As such, Pim-1 is an attractive therapeutic target. TP-3654 is a second-generation, oral Pim inhibitor currently in Phase I clinical trials in solid tumors and myelofibrosis (NCT03715504 and NCT04176198). TP-3654 inhibits all three Pim kinases, with Ki values for Pim-1 (5nM), and Pim-2 and Pim-3 l 2021 Apr 10-15 and May 17-21. Phi...

TNK1 (thirty-eight-negative kinase 1) is an ACK non-receptor tyrosine kinase expressed during developmental hematopoiesis. Expression in adult tissues has been implicated in hematologic malignancies and in mediation of cellular stress and apoptosis. A truncating mutation leading to overexpression and hyperactivation of TNK1 drives growth and proliferation of L540 cells, a Hodgkin9s lymphoma-derived cell line. TNK1 knockdown is known to reduce phosphorylation of downstream effectors STAT3 and STAT5 in L540 cells, to sensitize cancer cells to stress. However, there are no specific TNK1 inhibitors currently approved for use in hematological malignancies. We propose that pharmacological inhibition of TNK1 phosphorylation can suppress TNK1-driven pathology in preclinical tumor models. TP-5809 is a novel small molecule that potently inhibits TNK1 with an IC50 of 1.5 nM in a biochemical kinase assay. TP-5809 shows cellular target engagement at low nanomolar concentrations (IC50 = 11.3 nM) ...

This study investigated the influence of the cholinergic system on neuro-inflammation using nicotinic and muscarinic receptor agonists and antagonists. Intracerebroventricular (ICV) injection of lipopolysaccharide (LPS, 50 microg) was used to induce neuro-inflammation in rats and estimations of pro-inflammatory cytokines, alpha7 nicotinic acetylcholine receptor (nAChR) mRNA expression were done in striatum, cerebral cortex, hippocampus and hypothalamus at 24 h after LPS injection. Nicotine (0.2, 0.4 and 0.8 mg/kg, i.p.) or oxotremorine (0.2, 0.4 and 0.8 mg/kg, i.p.) were administered 2 h prior to sacrifice. We found that only nicotine was able to block the proinflammatory cytokines induced by LPS whereas, oxotremorine was found ineffective. Methyllycaconitine (MLA; 1.25, 2.5 and 5 mg/kg, i.p.), an alpha7 nAChR antagonist or dihydro-beta-erythroidine (DHbetaE; 1.25, 2.5 and 5 mg/kg, i.p.), an alpha4beta2 nAChR antagonist, was given 20 min prior to nicotine in LPS-treated rats. Methyl...

Quality nutrition during the period of brain formation is a predictor of brain functional capacity and plasticity during adulthood; however it is not clear how this conferred plasticity imparts long-term neural resilience. Here we report that early exposure to dietary omega-3 fatty acids orchestrates key interactions between metabolic signals and Bdnf methylation creating a reservoir of neuroplasticity that can protect the brain against the deleterious effects of switching to a Western diet (WD). We observed that the switch to a WD increased Bdnf methylation specific to exon IV, in proportion to anxiety-like behavior, in Sprague Dawley rats reared in low omega-3 fatty acid diet, and these effects were abolished by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. Blocking methylation also counteracted the reducing action of WD on the transcription regulator CTCF binding to Bdnf promoter IV. In vitro studies confirmed that CTCF binding to Bdnf promoter IV is essential for the action of DHA on BDNF regulation. Diet is also intrinsically associated to cell metabolism, and here we show that the switch to WD downregulated cell metabolism (NAD/NADH ratio and SIRT1). The fact that DNA methyltransferase inhibitor did not alter these parameters suggests they occur upstream to methylation. In turn, the methylation inhibitor counteracted the action of WD on PGC-1α, a mitochondrial transcription co-activator and BDNF regulator, suggesting that PGC-1α is an effector of Bdnf methylation. Results support a model in which diet can build an "epigenetic memory" during brain formation that confers resilience to metabolic perturbations occurring in adulthood.

In the present study, neuroinflammation was induced by bilateral intracerebroventricular (ICV) administration of Lipopolysaccharide (LPS). Proinflammatory cytokines (TNF-alpha and IL-1beta), acetylcholinesterase (AChE) activity, malondialdehyde (MDA) and reduced glutathione (GSH) were studied as markers for neuroinflammation, cholinergic activity and oxidative stress respectively in different brain regions at different time points after LPS injection. LPS produced increase in proinflammatory cytokines, MDA and the decrease in level of GSH at 24 h indicating a state of inflammation in brain regions, which was significantly blocked by Ibuprofen, a non steroidal anti-inflammatory drug. Enhanced AChE activity with these inflammatory markers after LPS administration indicates a possible relationship between neuroinflammation and cholinergic system during the development of neurodegenerative diseases.

Lipopolysaccharide (LPS) and rotenone induced oxidative stress was investigated in homogenates of rat brain regions - striatum, mid brain, frontal cortex and hippocampus. LPS at concentration 1, 25 and 50μg and rotenone 1, 2 and 4mM was incubated with the brain homogenates and caused decrease in reduced glutathione (GSH) and rise in malondialdehyde (MDA) in different brain regions but in a varied manner. Anti-oxidants melatonin and nimesulide (0.75, 1.5 and 3mM) were incubated concurrently with LPS (50μg) and rotenone (4mM) in the homogenates. Melatonin as well as nimesulide (3mM) suppressed the LPS and rotenone induced increase in MDA but their effect on GSH differed. Lack of uniform response by different brain areas to LPS, rotenone and antioxidants indicate that sensitivity to oxidative stress may differ among the brain areas; this variability in sensitivity may be of significance in relation to free radicals induced selective neuronal degeneration.

To discuss studies in humans and animals revealing the ability of foods to benefit the brain: new information with regards to mechanisms of action and the treatment of neurological and psychiatric disorders. Dietary factors exert their effects on the brain by affecting molecular events related to the management of energy metabolism and synaptic plasticity. Energy metabolism influences neuronal function, neuronal signaling, and synaptic plasticity, ultimately affecting mental health. Epigenetic regulation of neuronal plasticity appears as an important mechanism by which foods can prolong their effects on long-term neuronal plasticity. The prime focus of the discussion is to emphasize the role of cell metabolism as a mediator for the action of foods on the brain. Oxidative stress promotes damage to phospholipids present in the plasma membrane such as the omega-3 fatty acid docosahexenoic acid, disrupting neuronal signaling. Thus, dietary docosahexenoic acid seems crucial for supporting plasma membrane function, interneuronal signaling, and cognition. The dual action of brain-derived neurotrophic factor in neuronal metabolism and synaptic plasticity is crucial for activating signaling cascades under the action of diet and other environmental factors, using mechanisms of epigenetic regulation.

Basal forebrain cholinergic neurons and oxidative stress in brain have been suggested to play an important role in the regulation of memory functions. Therefore, the present study was planned to study the effect of donepezil, an anticholinesterase antidementia drug, insulin and melatonin, an antioxidant, on memory deficit and acetylcholinesterase (AChE) activity in brain areas of scopolamine-induced amnesic mice. Memory was tested by passive avoidance (PA) test in Swiss adult male mice. A significant increase in transfer latency time (TLT) in 2nd trial as compared to 1st trial is considered as successful learning. Scopolamine (3 mg/kg i.p.) was administered 5 min prior to 1st trial to induce amnesia. AChE activity in detergent soluble (DS) and salt soluble (SS) fractions was estimated in brain areas after completion of 2nd trial. Scopolamine was effective in producing memory impairment (amnesia) which was reverted by donepezil (5 mg/kg p.o.), insulin (1 IU/kg i.p.) and melatonin (20 mg/kg p.o.). AChE activity in DS fraction of scopolamine amnesic mice was inhibited by donepezil, insulin and melatonin with varying extent in different brain regions, whereas AChE activity in SS fraction was not much affected. The results demonstrate that anti-amnesic effect of donepezil, insulin and melatonin may be mediated through enhancement of cholinergic activity.

Metabolic dysfunction occurring after traumatic brain injury (TBI) is an important risk factor for the development of psychiatric illness. In the present study, we utilized an omega-3 diet during early life as a metabolic preconditioning to alter the course of TBI during adulthood. TBI animals under omega-3 deficiency were more prone to alterations in energy homeostasis (adenosine monophosphate-activated protein kinase; AMPK phosphorylation and cytochrome C oxidase II; COII levels) and mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PGC-1α and mitochondrial transcription factor A; TFAM). A similar response was found for brain-derived neurotrophic factor (BDNF) and its signaling through tropomyosin receptor kinase B (TrkB). The results from in vitro studies showed that 7,8-dihydroxyflavone (7,8-DHF), a TrkB receptor agonist, upregulates the levels of biogenesis activator PGC-1α, and CREB phosphorylation in neuroblastoma cells suggesting that BDNF-TrkB signaling is pivotal for engaging signals related to synaptic plasticity and energy metabolism. The treatment with 7,8-DHF elevated the mitochondrial respiratory capacity, which emphasizes the role of BDNF-TrkB signaling as mitochondrial bioenergetics stimulator. Omega-3 deficiency worsened the effects of TBI on anxiety-like behavior and potentiated a reduction of anxiolytic neuropeptide Y1 receptor (NPY1R). These results highlight the action of metabolic preconditioning for building long-term neuronal resilience against TBI incurred during adulthood. Overall, the results emphasize the interactive action of metabolic and plasticity signals for supporting neurological health.