A chemical approach facilitates CRISPRa-only human iPSC generation and minimizes the number of targeted loci required.

Aim: We explored the generation of human induced pluripotent stem cells (iPSCs) solely through the transcriptional activation of endogenous genes by CRISPR activation (CRISPRa). Methods: Minimal number of human-specific guide RNAs targeting a limited set of loci were used with a unique cocktail of small molecules (CRISPRa-SM). Results: iPSC clones were efficiently generated by CRISPRa-SM, expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. iPSCs showed genomic stability and robust pluripotent potential as assessed by in vitro and in vivo. Conclusion: CRISPRa-SM-generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine.

Combined chemical and CRISPRa-mediated approach leads to efficient generation of human iPSCs.

A chimeric antigen receptor uniquely recognizing MICA/B stress proteins provides an effective approach to target solid tumors.

BACKGROUND: The advent of chimeric antigen receptor (CAR) T cell therapies has transformed the treatment of hematological malignancies; however, broader therapeutic success of CAR T cells has been limited in solid tumors because of their frequently heterogeneous composition. Stress proteins in the MICA and MICB (MICA/B) family are broadly expressed by tumor cells following DNA damage but are rapidly shed to evade immune detection. METHODS: We have developed a novel CAR targeting the conserved α3 domain of MICA/B (3MICA/B CAR) and incorporated it into a multiplexed-engineered induced pluripotent stem cell (iPSC)-derived natural killer (NK) cell (3MICA/B CAR iNK) that expressed a shedding-resistant form of the CD16 Fc receptor to enable tumor recognition through two major targeting receptors. FINDINGS: We demonstrated that 3MICA/B CAR mitigates MICA/B shedding and inhibition via soluble MICA/B while simultaneously exhibiting antigen-specific anti-tumor reactivity across an expansive library of human cancer cell lines. Pre-clinical assessment of 3MICA/B CAR iNK cells demonstrated potent antigen-specific in vivo cytolytic activity against both solid and hematological xenograft models, which was further enhanced in combination with tumor-targeted therapeutic antibodies that activate the CD16 Fc receptor. CONCLUSIONS: Our work demonstrated 3MICA/B CAR iNK cells to be a promising multi-antigen-targeting cancer immunotherapy approach intended for solid tumors. FUNDING: Funded by Fate Therapeutics and NIH (R01CA238039).

Quadruple gene-engineered natural killer cells enable multi-antigen targeting for durable antitumor activity against multiple myeloma.

Allogeneic natural killer (NK) cell adoptive transfer is a promising treatment for several cancers but is less effective for the treatment of multiple myeloma. In this study, we report on quadruple gene-engineered induced pluripotent stem cell (iPSC)-derived NK cells designed for mass production from a renewable source and for dual targeting against multiple myeloma through the introduction of an NK cell-optimized chimeric antigen receptor (CAR) specific for B cell maturation antigen (BCMA) and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity when combined with therapeutic anti-CD38 antibodies. Additionally, these cells express a membrane-bound interleukin-15 fusion molecule to enhance function and persistence along with knock out of CD38 to prevent antibody-mediated fratricide and enhance NK cell metabolic fitness. In various preclinical models, including xenogeneic adoptive transfer models, quadruple gene-engineered NK cells consistently demonstrate durable antitumor activity independent of exogenous cytokine support. Results presented here support clinical translation of this off-the-shelf strategy for effective treatment of multiple myeloma.

Dual antigen-targeted off-the-shelf NK cells show durable response and prevent antigen escape in lymphoma and leukemia.

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition, we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies.

Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy.

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer.

iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy.

The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti-PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an "off-the-shelf" source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a "cold" tumor "hot" by promoting the influx of activated T cells to augment checkpoint inhibitor therapies.

A Single Mutation Traps a Half-Sites Reactive Enzyme in Midstream, Explaining Asymmetry in Hydride Transfer.

In Escherichia coli thymidylate synthase (EcTS), rate-determining hydride transfer from the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate to the intermediate 5-methylene-2'-deoxyuridine 5'-monophosphate occurs by hydrogen tunneling, requiring precise alignment of reactants and a closed binding cavity, sealed by the C-terminal carboxyl group. Mutations that destabilize the closed conformation of the binding cavity allow small molecules such as ß-mercaptoethanol (ß-ME) to enter the active site and compete with hydride for addition to the 5-methylene group of the intermediate. The C-terminal deletion mutant of EcTS produced the ß-ME adduct in proportions that varied dramatically with cofactor concentration, from 50% at low cofactor concentrations to 0% at saturating cofactor conditions, suggesting communication between active sites. We report the 2.4 Å X-ray structure of the C-terminal deletion mutant of E. coli TS in complex with a substrate and a cofactor analogue, CB3717. The structure is asymmetric, with reactants aligned in a manner consistent with hydride transfer in only one active site. In the second site, CB3717 has shifted to a site where the normal cofactor would be unlikely to form 5-methylene-2'-deoxyuridine 5'-monophosphate, consistent with no formation of the ß-ME adduct. The structure shows how the binding of the cofactor at one site triggers hydride transfer and borrows needed stabilization from substrate binding at the second site. It indicates pathways through the dimer interface that contribute to allostery relevant to half-sites reactivity.

Cardiotrophin 1 stimulates beneficial myogenic and vascular remodeling of the heart.

The post-natal heart adapts to stress and overload through hypertrophic growth, a process that may be pathologic or beneficial (physiologic hypertrophy). Physiologic hypertrophy improves cardiac performance in both healthy and diseased individuals, yet the mechanisms that propagate this favorable adaptation remain poorly defined. We identify the cytokine cardiotrophin 1 (CT1) as a factor capable of recapitulating the key features of physiologic growth of the heart including transient and reversible hypertrophy of the myocardium, and stimulation of cardiomyocyte-derived angiogenic signals leading to increased vascularity. The capacity of CT1 to induce physiologic hypertrophy originates from a CK2-mediated restraining of caspase activation, preventing the transition to unrestrained pathologic growth. Exogenous CT1 protein delivery attenuated pathology and restored contractile function in a severe model of right heart failure, suggesting a novel treatment option for this intractable cardiac disease.

KLYP956 is a non-imidazole-based orally active inhibitor of nitric-oxide synthase dimerization.

Nitric-oxide synthases (NOS) generate nitric oxide (NO) through the oxidation of l-arginine. Inappropriate or excessive production of NO by NOS is associated with the pathophysiology of various disease states. Efforts to treat these disorders by developing arginine mimetic, substrate-competitive NOS inhibitors as drugs have met with little success. Small-molecule-mediated inhibition of NOS dimerization represents an intriguing alternative to substrate-competitive inhibition. An ultra-high-throughput cell-based screen of 880,000 small molecules identified a novel quinolinone with inducible NOS (iNOS) inhibitory activity. Exploratory chemistry based on this initial screening hit resulted in the synthesis of KLYP956, which inhibits iNOS at low nanomolar concentrations. The iNOS inhibitory potency of KLYP956 is insensitive to changes in concentrations of the substrate arginine, or the cofactor tetrahydrobiopterin. Mechanistic analysis suggests that KLYP956 binds the oxygenase domain in the vicinity of the active site heme and inhibits iNOS and neuronal NOS (nNOS) by preventing the formation of enzymatically active dimers. Oral administration of KLYP956 [N-(3-chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-4-methylthiazole-5-carboxamide] inhibits iNOS activity in a murine model of endotoxemia and blocks pain behaviors in a formalin model of nociception. KLYP956 thus represents the first nonimidazole-based inhibitor of iNOS and nNOS dimerization and provides a novel pharmaceutical alternative to previously described substrate competitive inhibitors.

Structure of a TrmA-RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m5U methyltransferases.

TrmA catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of U54 in most tRNAs. We solved the structure of the Escherichia coli 5-methyluridine (m(5)U) 54 tRNA methyltransferase (MTase) TrmA in a covalent complex with a 19-nt T arm analog to 2.4-A resolution. Mutation of the TrmA catalytic base Glu-358 to Gln arrested catalysis and allowed isolation of the covalent TrmA-RNA complex for crystallization. The protein-RNA interface includes 6 nt of the T loop and two proximal base pairs of the stem. U54 is flipped out of the loop into the active site. A58 occupies the space of the everted U54 and is part of a collinear base stack G53-A58-G57-C56-U55. The RNA fold is different from T loop conformations in unbound tRNA or T arm analogs, but nearly identical to the fold of the RNA loop bound at the active site of the m(5)U MTase RumA. In both enzymes, this consensus fold presents the target U and the following two bases to a conserved binding groove on the protein. Outside of this fold, the RumA and TrmA substrates have completely different structures and protein interfaces. Loop residues other than the target U54 make more than half of their hydrogen bonds to the protein via sugar-phosphate moieties, accounting, in part, for the broad consensus sequence for TrmA substrates.

Synthesis and SAR of thiophene containing kinesin spindle protein (KSP) inhibitors.

We have identified and synthesized a series of thiophene containing inhibitors of kinesin spindle protein. SAR studies led to the synthesis of 33, which was co-crystallized with KSP and determined to bind to an allosteric pocket previously described for other known KSP inhibitors.

The role of protein dynamics in thymidylate synthase catalysis: variants of conserved 2'-deoxyuridine 5'-monophosphate (dUMP)-binding Tyr-261.

The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.

A unique RNA Fold in the RumA-RNA-cofactor ternary complex contributes to substrate selectivity and enzymatic function.

A single base (U1939) within E. coli 23S ribosomal RNA is methylated by its dedicated enzyme, RumA. The structure of RumA/RNA/S-adenosylhomocysteine uncovers the mechanism for achieving unique selectivity. The single-stranded substrate is "refolded" on the enzyme into a compact conformation with six key intra-RNA interactions. The RNA substrate contributes directly to catalysis. In addition to the target base, a second base is "flipped out" from the core loop to stack against the adenine of the cofactor S-adenosylhomocysteine. Nucleotides in permuted sequence order are stacked into the site vacated by the everted target U1939 and compensate for the energetic penalty of base eversion. The 3' hairpin segment of the RNA binds distal to the active site and provides binding energy that contributes to enhanced catalytic efficiency. Active collaboration of RNA in catalysis leads us to conclude that RumA and its substrate RNA may reflect features from the earliest RNA-protein era.

Crystal structure of RumA, an iron-sulfur cluster containing E. coli ribosomal RNA 5-methyluridine methyltransferase.

RumA catalyzes transfer of a methyl group from S-adenosylmethionine (SAM) specifically to uridine 1939 of 23S ribosomal RNA in Escherichia coli to yield 5-methyluridine. We determined the crystal structure of RumA at 1.95 A resolution. The protein is organized into three structural domains: The N-terminal domain contains sequence homology to the conserved TRAM motif and displays a five-stranded beta barrel architecture characteristic of an oligosaccharide/oligonucleotide binding fold. The central domain contains a [Fe(4)S(4)] cluster coordinated by four conserved cysteine residues. The C-terminal domain displays the typical SAM-dependent methyltransferase fold. The catalytic nucleophile Cys389 lies in a motif different from that in DNA 5-methylcytosine methyltransferases. The electrostatic potential surface reveals a predominately positively charged area that covers the concave surface of the first two domains and suggests an RNA binding mode. The iron-sulfur cluster may be involved in the correct folding of the protein or may have a role in RNA binding.