Pharmacological Characterization of IW-1973, a Novel Soluble Guanylate Cyclase Stimulator with Extensive Tissue Distribution, Antihypertensive, Anti-Inflammatory, and Antifibrotic Effects in Preclinical Models of Disease.

Soluble guanylate cyclase (sGC), a key signal-transduction enzyme, increases the conversion of guanosine-5'-triphosphate to cGMP upon binding of nitric oxide (NO). Endothelial dysfunction and/or reduced NO signaling have been implicated in cardiovascular disease pathogenesis and complications of diabetes and have been associated with other disease states and aging. Soluble guanylate cyclase (sGC) stimulators are small-molecule drugs that bind sGC and enhance NO-mediated cGMP signaling. The pharmacological characterization of IW-1973 [1,1,1,3,3,3-hexafluoro-2-(((5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl) pyrimidin-4-yl)amino)methyl)propan-2-ol], a novel clinical-stage sGC stimulator under clinical investigation for treatment of heart failure with preserved ejection fraction and diabetic nephropathy, is described. In the presence of NO, IW-1973 stimulated sGC in a human purified enzyme assay and a HEK-293 whole cell assay. sGC stimulation by IW-1973 in cells was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. IW-1973, at doses of 1-10 mg/kg, significantly lowered blood pressure in normotensive and spontaneously hypertensive rats. In a Dahl salt-sensitive hypertension model, IW-1973 significantly reduced blood pressure, inflammatory cytokine levels, and renal disease markers, including proteinuria and renal fibrotic gene expression. The results were affirmed in mouse lipopolysaccharide-induced inflammation and rat unilateral ureteral obstruction renal fibrosis models. A quantitative whole-body autoradiography study of IW-1973 revealed extensive tissue distribution and pharmacokinetic studies showed a large volume of distribution and a profile consistent with predicted once-a-day dosing in humans. In summary, IW-1973 is a potent, orally available sGC stimulator that exhibits renoprotective, anti-inflammatory, and antifibrotic effects in nonclinical models.

AID-initiated DNA lesions are differentially processed in distinct B cell populations.

Activation-induced deaminase (AID) initiates U:G mismatches, causing point mutations or DNA double-stranded breaks at Ig loci. How AID-initiated lesions are prevented from inducing genome-wide damage remains elusive. A differential DNA repair mechanism might protect certain non-Ig loci such as c-myc from AID attack. However, determinants regulating such protective mechanisms are largely unknown. To test whether target DNA sequences modulate protective mechanisms via altering the processing manner of AID-initiated lesions, we established a knock-in model by inserting an Sγ2b region, a bona fide AID target, into the first intron of c-myc. Unexpectedly, we found that the inserted S region did not mutate or enhance c-myc genomic instability, due to error-free repair of AID-initiated lesions, in Ag-stimulated germinal center B cells. In contrast, in vitro cytokine-activated B cells display a much higher level of c-myc genomic instability in an AID- and S region-dependent manner. Furthermore, we observe a comparable frequency of AID deamination events between the c-myc intronic sequence and inserted S region in different B cell populations, demonstrating a similar frequency of AID targeting. Thus, our study reveals a clear difference between germinal center and cytokine-activated B cells in their ability to develop genomic instability, attributable to a differential processing of AID-initiated lesions in distinct B cell populations. We propose that locus-specific regulatory mechanisms (e.g., transcription) appear to not only override the effects of S region sequence on AID targeting frequency but also influence the repair manner of AID-initiated lesions.

Chromosomal location targets different MYC family gene members for oncogenic translocations.

The MYC family of cellular oncogenes includes c-Myc, N-myc, and L-myc, which encode transcriptional regulators involved in the control of cell proliferation and death. Accordingly, these genes become aberrantly activated and expressed in specific types of cancers. For example, c-Myc translocations occur frequently in human B lymphoid tumors, while N-myc gene amplification is frequent in human neuroblastomas. The observed association between aberrations in particular MYC family genes and specific subsets of malignancies might reflect, at least in part, tissue-specific differences in expression or function of a given MYC gene. Since c-Myc and N-myc share substantial functional redundancy, another factor that could influence tumor-specific gene activation would be mechanisms that target aberrations (e.g., translocations) in a given MYC gene in a particular tumor progenitor cell type. We have previously shown that mice deficient for the DNA Ligase4 (Lig4) nonhomologous DNA end-joining factor and the p53 tumor suppressor routinely develop progenitor (pro)-B cell lymphomas that harbor translocations leading to c-Myc amplification. Here, we report that a modified allele in which the c-Myc coding sequence is replaced by N-myc coding sequence (NCR allele) competes well with the wild-type c-Myc allele as a target for oncogenic translocations and amplifications in the Lig4/p53-deficient pro-B cell lymphoma model. Tumor onset, type, and cytological aberrations are similar in tumors harboring either the wild-type c-Myc gene or the NCR allele. Our results support the notion that particular features of the c-Myc locus select it as a preferential translocation/amplification target, compared to the endogenous N-myc locus, in Lig4/p53-deficient pro-B cell lymphomas.

STimulator of INterferon Genes Agonism Accelerates Antitumor Activity in Poorly Immunogenic Tumors.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.

The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation.

Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID-RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.

ILT3 (LILRB4) Promotes the Immunosuppressive Function of Tumor-Educated Human Monocytic Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion. IMPLICATIONS: These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.

Discovery of Diaminopyrimidine Carboxamide HPK1 Inhibitors as Preclinical Immunotherapy Tool Compounds.

Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase, is a negative immune regulator of T cell receptor (TCR) and B cell signaling that is primarily expressed in hematopoietic cells. Accordingly, it has been reported that HPK1 loss-of-function in HPK1 kinase-dead syngeneic mouse models shows enhanced T cell signaling and cytokine production as well as tumor growth inhibition in vivo, supporting its value as an immunotherapeutic target. Herein, we present the structurally enabled discovery of novel, potent, and selective diaminopyrimidine carboxamide HPK1 inhibitors. The key discovery of a carboxamide moiety was essential for enhanced enzyme inhibitory potency and kinome selectivity as well as sustained elevation of cellular IL-2 production across a titration range in human peripheral blood mononuclear cells. The elucidation of structure-activity relationships using various pendant amino ring systems allowed for the identification of several small molecule type-I inhibitors with promising in vitro profiles.

Development of High-Throughput Assays for Evaluation of Hematopoietic Progenitor Kinase 1 Inhibitors.

Hematopoietic progenitor kinase 1 (HPK1), also referred to as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine kinase that negatively regulates T-cell signaling by phosphorylating Ser376 of Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), a critical mediator of T-cell receptor activation. HPK1 loss of function mouse models demonstrated enhanced immune cell activation and beneficial antitumor activity. To enable discovery and functional characterization of high-affinity small-molecule HPK1 inhibitors, we have established high-throughput biochemical, cell-based, and novel pharmacodynamic (PD) assays. Kinase activity-based time-resolved fluorescence energy transfer (TR-FRET) assays were established as the primary biochemical approach to screen for potent inhibitors and assess selectivity against members of MAP4K and other closely related kinases. A proximal target engagement (TE) assay quantifying pSLP-76 levels as a readout and a distal assay measuring IL-2 secretion as a functional response were established using human peripheral blood mononuclear cells (PBMCs) from two healthy donors. Significant correlations between biochemical and cellular assays as well as excellent correlation between the two donors for the cellular assays were observed. pSLP-76 levels were further used as a PD marker in the preclinical murine model. This effort required the development of a novel ultrasensitive single-molecule array (SiMoA) assay to monitor pSLP-76 changes in mouse spleen.

Combination of EP4 antagonist MF-766 and anti-PD-1 promotes anti-tumor efficacy by modulating both lymphocytes and myeloid cells.

Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2-mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

Pharmacological inhibition of hematopoietic progenitor kinase 1 positively regulates T-cell function.

Hematopoietic progenitor kinase 1 (HPK1), a hematopoietic cell-specific Ste20-related serine/threonine kinase, is a negative regulator of signal transduction in immune cells, including T cells, B cells, and dendritic cells (DCs). In mice, HPK1 deficiency subverts inhibition of the anti-tumor immune response and is associated with functional augmentation of anti-tumor T cells. We have used a potent, small molecule HPK1 inhibitor, Compound 1, to investigate the effects of pharmacological intervention of HPK1 kinase activity in immune cells. Compound 1 enhanced Th1 cytokine production in T cells and fully reverted immune suppression imposed by the prostaglandin E2 (PGE2) and adenosine pathways in human T cells. Moreover, the combination of Compound 1 with pembrolizumab, a humanized monoclonal antibody against the programmed cell death protein 1 (PD-1), demonstrated a synergistic effect, resulting in enhanced interferon (IFN)-γ production. Collectively, our results suggest that blocking HPK1 kinase activity with small molecule inhibitors alone or in combination with checkpoint blockade may be an attractive approach for the immunotherapy of cancer.

Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo.

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

Productive coupling of accessible Vbeta14 segments and DJbeta complexes determines the frequency of Vbeta14 rearrangement.

To elucidate mechanisms that regulate Vbeta rearrangement, we generated and analyzed mice with a V(D)J recombination reporter cassette of germline Dbeta-Jbeta segments inserted into the endogenous Vbeta14 locus (Vbeta14(Rep)). As a control, we first generated and analyzed mice with the same Dbeta-Jbeta cassette targeted into the generally expressed c-myc locus (c-myc(Rep)). Substantial c-myc(Rep) recombination occurred in both T and B cells and initiated concurrently with endogenous Dbeta to Jbeta rearrangements in thymocytes. In contrast, Vbeta14(Rep) recombination was restricted to T cells and initiated after endogenous Dbeta to Jbeta rearrangements, but concurrently with endogenous Vbeta14 rearrangements. Thus, the local chromatin environment imparts lineage and developmental stage-specific accessibility upon the inserted reporter. Although Vbeta14 rearrangements occur on only 5% of endogenous TCRbeta alleles, the Vbeta14(Rep) cassette underwent rearrangement on 80-90% of alleles, supporting the suggestion that productive coupling of accessible Vbeta14 segments and DJbeta complexes influence the frequency of Vbeta14 rearrangements. Strikingly, Vbeta14(Rep) recombination also occurs on TCRbeta alleles lacking endogenous Vbeta to DJbeta rearrangements, indicating that Vbeta14 accessibility per se is not subject to allelic exclusion.

Vbeta cluster sequences reduce the frequency of primary Vbeta2 and Vbeta14 rearrangements.

T-cell receptor (TCR) beta variable region exons are assembled from numerous gene segments in a highly ordered and regulated manner. To elucidate mechanisms and identify cis-acting elements that control Vbeta rearrangement, we generated an endogenous TCR-beta allele with only the Vbeta2, Vbeta4, and Vbeta14 segments. We found that alphabeta T lineage cells containing this Vbeta(2-4-14) allele and a wild-type TCR-beta allele developed normally, but exhibited a significant increase in Vbeta2(+) and Vbeta14(+) cells. To quantify Vbeta rearrangements on the Vbeta(2-4-14) allele, we generated alphabeta T-cell hybridomas and analyzed TCR-beta rearrangements. Despite the deletion of almost all Vbeta segments and 234 kb of Vbeta cluster sequences, the Vbeta(2-4-14) allele exhibited only a slight decrease in Vbeta rearrangement as compared with the wild-type TCR-beta allele. Thus, cis-acting control elements essential for directing Vbeta rearrangement across large chromosomal distances are not located within the Vbeta cluster. We also found a significant increase in the frequency of Vbeta rearrangements involving Vbeta2 and Vbeta14, but not Vbeta4, on the Vbeta(2-4-14) allele. Collectively, our data suggest that Vbeta cluster sequences reduce the frequency of Vbeta2 and Vbeta14 rearrangements by competing with the productive coupling of accessible Vbeta2 and Vbeta14 segments with DJbeta1 complexes.

Aberrant V(D)J recombination is not required for rapid development of H2ax/p53-deficient thymic lymphomas with clonal translocations.

Histone H2AX is required to maintain genomic stability in cells and to suppress malignant transformation of lymphocytes in mice. H2ax(-/-)p53(-/-) mice succumb predominantly to immature alphabeta T-cell lymphomas with translocations, deletions, and genomic amplifications that do not involve T-cell receptor (TCR). In addition, H2ax(-/-)p53(-/-) mice also develop at lower frequencies B and T lymphomas with antigen receptor locus translocations. V(D)J recombination is initiated through the programmed induction of DNA double-strand breaks (DSBs) by the RAG1/RAG2 endonuclease. Because promiscuous RAG1/RAG2 cutting outside of antigen receptor loci can promote genomic instability, H2ax(-/-)p53(-/-) T-lineage lymphomas might arise, at least in part, through erroneous V(D)J recombination. Here, we show that H2ax(-/-)p53(-/-)Rag2(-/-) mice exhibit a similar genetic predisposition as do H2ax(-/-)p53(-/-) mice to thymic lymphoma with translocations, deletions, and amplifications. We also found that H2ax(-/-)p53(-/-)Rag2(-/-) mice often develop thymic lymphomas with loss or deletion of the p53(+) locus. Our data show that aberrant V(D)J recombination is not required for rapid onset of H2ax/p53-deficient thymic lymphomas with genomic instability and that H2ax deficiency predisposes p53(-/-)Rag2(-/-) thymocytes to transformation associated with p53 inactivation. Thus, H2AX is essential for suppressing the transformation of developing thymocytes arising from the aberrant repair of spontaneous DSBs.

Restriction of endogenous T cell antigen receptor beta rearrangements to Vbeta14 through selective recombination signal sequence modifications.

T cell antigen receptor (TCR)beta V(D)J variable region exon assembly is ordered, with Dbeta to Jbeta rearrangements occurring before joining of Vbetas to a DJbeta complex. Germ-line V(D)J segments are flanked by recombination signal (RS) sequences, which consist of heptamers and nonamers separated by a spacer of 12 (12-RS) or 23 (23-RS) bp. V(D)J recombination is restricted by the 12/23 rule; joining occurs only between gene segments flanked by 12-RSs and 23-RSs. Vbeta segments have 23-RSs and Jbeta segments 12-RSs, which based on the 12/23 rule should allow direct joining. However, Vbeta segments rearrange only to DJbeta complexes and not Jbeta segments, because of restrictions beyond 12/23 (B12/23) that make the Vbeta23-RS incompatible with the Jbeta12-RS. To determine whether direct Vbeta to Jbeta joining occurs if flanking RSs are B12/23 compatible, we generated mice whose lymphocytes contained replacement of the Vbeta1412-RS with the 3'Dbeta112-RS on a TCRbeta allele lacking Dbeta segments (the Jbeta1(M6) allele). Mice heterozygous for the Jbeta1(M6) allele had dramatically increased Vbeta14(+) thymocyte and T cell numbers and decreased numbers of cells expressing other Vbetas. This altered Vbeta repertoire resulted from direct Vbeta14 to Jbeta1 rearrangements on the Jbeta1(M6) allele. Mice harboring lymphocytes homozygous for Jbeta1(M6) allele developed normal thymocyte and T cell numbers with all expressing Vbeta14. Our findings show that selective RS modifications enforce rearrangement of a specific Vbeta gene segment and demonstrate the importance of B12/23 mechanisms for ensuring generation of diverse TCRbeta repertoires.