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Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.

The serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy. To assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both and . In a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. , STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti-PD-L1 antibody in the MCA205 model. In summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses and . This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy.

BackgroundCheckpoint immunotherapies have revolutionized solid tumor treatment yet durably benefit a minority of patients, as they rely on endogenous anti-tumor T cells. A potential solution for patients lacking functional endogenous anti-tumor T cells is engineering their T cells with exogenous T cell receptors (TCRs) to target and kill tumor cells. Initial clinical trials with TCR engineered T cell therapies (TCR-Ts) produced partial, short-lasting responses because they targeted single tumor antigens. Solid tumors are notoriously heterogenous with highly variable antigen expression. Recent discoveries also identified HLA loss of heterozygosity occurring in up to 40% of solid tumors, allowing tumor cells to evade T cell attack.To overcome this heterogeneity, TScan has developed T-Plex, a multiplexed cell therapy comprising 2–3 different TCR-Ts, chosen from a collection of TCR-Ts called the ImmunoBank, to target different tumor antigens on different HLA types with confirmed tumor expression. To deepen clinical responses, TCR-T cells are engineered to express CD8α/β co-receptors that, in preclinical experiments, enable CD4+ helper T cells to have >100-fold improved cytotoxicity and cytokine secretion. Finally, to allow T cell persistence despite immunosuppressive TGF-β in tumor microenvironments, TCR-T cells also express the dominant negative TGF-β receptor, enabling ~10-fold improved proliferation despite the presence of TGF-β. These additional genes can be included because of a proprietary transposon vector with larger cargo limit.MethodsThe Phase 1 study utilizes a separate screening protocol to identify patients any time during standard clinical care, enabling rapid enrollment into the treatment protocol upon disease progression. Screening comprises germline HLA testing, then archival tumor testing for antigen expression and HLA loss. Treatment includes standard lymphodepletion followed by one or 2 doses of T-Plex infused 28 days apart. Dose escalation starts with testing single TCR-Ts in dose levels 1 and 2. Thereafter, TCR-Ts are combined and escalated in dose levels 3 and 4. TCR-Ts initially in the master protocol target MAGE-A1 or HPV16 on HLA-A*02:01 or MAGE-A1 on HLA-C*07:02. Additional TCR-Ts added to the ImmunoBank and master protocol go through dose levels 1 and 2 as single therapies before becoming available for multiplexed dose levels 3 and 4. Primary endpoints include safety and feasibility, secondary endpoints are rates and durations of response and exploratory endpoints measure T cell persistence. Three additional TCR-Ts are on track to be added to the ImmunoBank in 2023, that could allow 50–80% of common solid tumor patients to qualify for multiplexed TCR-T therapy.Trial RegistrationNCT05812027Ethics ApprovalThe study obtained ethics approval from WCG-IRB (20230668 and 20230670). Participants give/will give informed consent before taking part.

BackgroundEngineered T cell therapy holds great promise for treating solid tumors. To date, clinical investigations of TCR-T cell therapies have targeted one antigen/HLA at a time and have produced encouraging but partial response rates with limited durations. While heterogeneity of antigen expression is appreciated as a likely driver of patient relapse, the contribution of HLA loss of heterozygosity (LOH), occurring in up to 40% of tumors, is only now gaining attention. To address both antigen heterogeneity and HLA LOH requires a collection of TCRs recognizing multiple targets presented on multiple HLAs. MAGE-A1 is a cancer-testes antigen previously identified as the target of expanded tumor infiltrating T-cells using TScan’s screening technology. Currently, TScan has two MAGE-A1-TCR-T products, recognizing epitopes on A*02:01 and C*07:02 approved for clinical development. Here we report discovery and lead selection of a MAGE-A1 TCR recognizing an epitope on A*01:01 (~24% population frequency).MethodsWe discovered TCRs specific for an A*01:01- restricted MAGE-A1-derived epitope using TScan’s proprietary ReceptorScan platform. Using an activation-based screening technology termed ActivScan, we screened a library of MAGE-A1-specific TCRs to select for greatest avidity and expression. These TCRs were functionally characterized using a panel of MAGE-A1 expressing A*01:01-positive cell lines and a xenograft mouse model. Lead TCRs were assessed for potential off-target reactivity using our proprietary SafetyScan platform, which evaluates recognition of antigens from all proteins that comprise the human proteome. Safety was further evaluated by examining alloreactivity to high-frequency Class I HLAs and by testing TCR reactivity to normal primary human cells and cell lines.ResultsReceptorScan identified 1181 TCRs specific for the MAGE-A1 A*01:01 epitope. Following selection of high-expressing and high avidity MAGE-A1-specific TCRs in ActivScan, 14 TCRs were evaluated for their cytotoxic function, and 5 TCRs compared favorably to a clinical-stage benchmark TCR for cytotoxicity and cytokine release. Safety assessment demonstrated that few putative off-target peptides were recognized, minimal alloreactivity was observed to 110 allotypes tested, and no reactivity to target-negative cell lines were observed.ConclusionsA novel HLA-A*01:01 restricted TCR-T cell therapy candidate has advanced to pre-clinical studies. Addition of this product to TScan’s ImmunoBank (collection of TCRs) would extend MAGE-A1 TCR-T therapy for solid tumors on three different HLA alleles, potentially expanding the addressable patient population. Importantly, this creates a unique opportunity to simultaneously target tumor antigens presented on HLA alleles on different chromosomes, thus circumventing tumor evasion by HLA LOH with the goal of improving patient responses.Ethics ApprovalThe Testing facility specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care.The animal care and use program at Explora, now CR Discovery Services, is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals. IACUC number: EB17–010-301.

Hyer et al . generate a potent and specific small-molecule inhibitor of the E1 ubiquitin-activating enzyme UBE1 that has antitumor activity in mice against a wide variety of tumor types. The ubiquitin–proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin–proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer. Targeting ubiquitin The ubiquitin–proteasome system tags intra-cellular proteins for degradation as part of the regulatory regime for many cellular functions, some cancer related. Proteasome inhibitors are now emerging as antitumour drugs, the first in clinical use being bortezomib, for the treatment of multiple myeloma and some lymphomas. Soucy et al . now report the discovery of MLN4924, a small molecule inhibitor of NEDD8-activating enzyme (NAE), now in phase I clinical trials. NAE regulates the activity of a subtype of ubiquitin ligases, the cullin-RING, which in turn controls the degradation of various cellular proteins. MLN4924 induces cancer cell death and exerts antitumour activity in mouse cancer models. This study has developed the first small molecule NEDD8-activating enzyme (NAE) inhibitor, which induces cancer cell death and exerts anti-tumour activity in preclinical mouse models. This work establishes NAE as an anti-cancer target and may lead to new anti-cancer therapeutics.

BackgroundT-Plex is an autologous TCR-T cell therapy product comprising customized combinations of 2–3 TCR-T cell components that recognize different tumor antigens presented on specific HLA class I molecules. Each component of T-Plex is engineered using a transposon-based vector encoding the therapeutic TCR, CD8α and CD8β co-receptors, a CD34 epitope tag, a dominant-negative TGFβRII (DN-TGFβRII), and a mutated form of dihydrofolate reductase (DHFRdm). TSC-200-A0201 is intended for the treatment of HPV16+ HLA-A*02:01+ cancers. HPV16 is an oncogenic virus responsible for ~57% of cervical cancers and ~21% of head and neck squamous cell carcinomas. HPV16 E7 oncoprotein drives oncogenic transformation of infected cells and is not expressed by healthy tissues, making it a compelling immunotherapeutic target.MethodsThe TSC-200-A0201 TCR is a naturally occurring TCR discovered using TScan’s proprietary ReceptorScan platform. TSC-200-A0201 TCR-T cells engineered using a full-scale representative workflow for the planned manufacturing process were used to investigate the in-vitro pharmacology and toxicology of TSC-200-A0201. TSC-200-A0201 was evaluated for avidity and target-dependent cytotoxicity, proliferation, and cytokine secretion in vitro as well as for anti-tumor efficacy in vivo. The contribution of DN-TGFβRII was assessed by testing the ability of TSC-200-A0201 TCR-T cells to resist the immuno-suppressive effects of TGFβ. Further, TSC-200-A0201 was assessed for risk of alloreactivity and off-target recognition using TScan’s SafetyScan screen. Finally, to assess the risk of off-target/off-tumor activity, TSC-200-A0201 TCR-T cells were tested for their reactivity to an extensive panel of 74 healthy human primary and iPSC-derived cells from tissues that are traditionally assessed in toxicology studies, including those expressing high levels of the putative off-targets of TSC-200-A0201.ResultsTSC-200-A0201 displayed high avidity for the cognate peptide (EC50 of ~4.2 pg/mL) and target-dependent secretion of inflammatory cytokines, killing of target cells, and proliferation of both engineered CD4+ and CD8+ T cells. Moreover, TSC-200-A0201 successfully controlled the growth of HLA-A*02:01+ HPV16+ tumors in mice. Target-dependent IFN-γ production and proliferation of TSC-200-A0201 was maintained in the presence of physiological levels of TGFβ. Further, TSC-200-A0201 displayed no alloreactivity to the 110 most common class I HLAs in the US population. Although a few putative off-targets were identified in the SafetyScan screen, TSC-200-A0201 showed no reactivity to normal primary or iPSC-derived cells.ConclusionsTSC-200-A0201 exhibits high specificity and potency. Based on these results, TSC-200-A0201 has been cleared by the U.S. FDA for clinical development and has been incorporated in the T-Plex Phase 1 clinical trial master protocol.Ethics ApprovalAnimal studies were performed at CR Discovery Services, a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). This facility complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. IACUC number: EB17–010-301.