PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Furthermore, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5, and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1, and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a potential role of PTIP in the regulation of gene expression.

The MLL family of histone methyltransferases maintains active chromatin domains by methylating histone H3 on lysine 4 (H3K4). How MLL complexes recognize specific chromatin domains in a temporal and tissue-specific manner remains unclear. We show that the DNA-binding protein PAX2 promotes assembly of an H3K4 methyltransferase complex through the ubiquitously expressed nuclear factor PTIP (pax transcription activation domain interacting protein). PTIP copurifies with ALR, MLL3, and other components of a histone methyltransferase complex. PTIP promotes assembly of the ALR complex and H3K4 methylation at a PAX2-binding DNA element. Without PTIP, Pax2 binds to this element but does not assemble the ALR complex. Embryonic lethal ptip-null mutants and conditional mutants both show reduced levels of methylated H3K4. Thus, PTIP bridges DNA-binding developmental regulators to histone methyltransferase-dependent epigenetic regulation.

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The RET receptor tyrosine kinase is activated by GDNF and controls outgrowth and invasion of the ureteric bud epithelia in the developing kidney. In renal epithelial cells and in enteric neuronal precursor cells, activation of RET results in chemotaxis as Ret expressing cells invade the surrounding GDNF expressing tissue. One potential downstream signaling pathway governing RET mediated chemotaxis may require phosphatidylinositol 3-kinase (PI3K), which generates PI(3,4,5) triphosphate. The PTEN tumor suppressor gene encodes a protein and lipid phosphatase that regulates cell growth, apoptosis and many other cellular processes. PTEN helps regulate cellular chemotaxis by antagonizing the PI3K signaling pathway through dephosphorylation of phosphotidylinositol triphosphates. In this report, we show that PTEN suppresses RET mediated cell migration and chemotaxis in cell culture assays, that RET activation results in asymmetric localization of inositol triphosphates and that loss of PTEN affects the pattern of branching morphogenesis in developing mouse kidneys. These data suggest a critical role for the PI3K/PTEN axis in shaping the pattern of epithelial branches in response to RET activation.

Methylation of specific lysine residues in core histone proteins is essential for embryonic development and can impart active and inactive epigenetic marks on chromatin domains. The ubiquitous nuclear protein PTIP is encoded by the Paxip1 gene and is an essential component of a histone H3 lysine 4 (H3K4) methyltransferase complex conserved in metazoans. In order to determine if PTIP and its associated complexes are necessary for maintaining stable gene expression patterns in a terminally differentiated, non-dividing cell, we conditionally deleted PTIP in glomerular podocytes in mice. Renal development and function were not impaired in young mice. However, older animals progressively exhibited proteinuria and podocyte ultra structural defects similar to chronic glomerular disease. Loss of PTIP resulted in subtle changes in gene expression patterns prior to the onset of a renal disease phenotype. Chromatin immunoprecipitation showed a loss of PTIP binding and lower H3K4 methylation at the Ntrk3 (neurotrophic tyrosine kinase receptor, type 3) locus, whose expression was significantly reduced and whose function may be essential for podocyte foot process patterning. These data demonstrate that alterations or mutations in an epigenetic regulatory pathway can alter the phenotypes of differentiated cells and lead to a chronic disease state.

Introduction: Current therapies for multiple myeloma (MM), such as immunomodulatory agents, proteasome inhibitors, stem-cell transplantation, and monoclonal antibodies against tumor-associated antigens have greatly improved patient survival. However, MM remains an incurable disease as most patients will eventually relapse. Recent advances in targeted T-cell therapies have shown promise in clinical trials but the adaptive immune system may be insufficient to eradicate all MM clones. In contrast, treatments harnessing the innate immune system have been relatively underdeveloped in MM despite evidence suggesting a role of innate immunity in the efficacy of existing therapies. Innate or innate-like cells, such as NK and γδ T cells, have the potential to display strong anti-tumor activity, and strategies aimed to improve or re-direct their cytotoxicity represent a new opportunity for cancer immunotherapies and a complementary approach to existing therapies. Here we describe the preclinical characterization of CTX-8573, a novel multispecific antibody that targets B-cell maturation antigen (BCMA) on tumor cells and promotes potent cytotoxicity by NK and γδ T cells through engagement of the activating receptors NKp30 and CD16a. Method: Bispecific constructs were generated by appending two common-light chain compatible anti-NKp30 Fab fragments to the C-terminus of an anti-BCMA IgG1 antibody containing an afucosylated Fc for enhanced CD16a engagement. To test the effects of targeting NKp30 alone, variants were expressed with an aglycosylated Fc to eliminate CD16a binding. In-vitro assays were performed with primary NK or γδ T cells to determine innate-cell activation, cytokine production, proliferation, and target-cell cytotoxicity against tumor cell lines with a range of BCMA expression levels. In-vivo efficacy studies were performed in multiple humanized mouse models and pharmacokinetics and safety were evaluated in Cynomolgus monkeys. Results: CTX-8573 is highly expressed in CHO cells with minimal aggregation and displays stability, solubility, and binding to BCMA and NKp30 equivalent to the parental monoclonal antibodies. By engaging NKp30 and CD16a, CTX-8573 promotes potent cytotoxicity of BCMA expressing target cells by NK and γδ T cells with >100 fold reduced EC50 compared to the corresponding BCMA monoclonal antibody control. CTX-8573 also demonstrates robust killing of low BCMA expressing cell lines including RPMI-8226 where monoclonal BCMA antibodies lack activity. An aglycosylated variant of CTX-8573 lacking CD16a binding maintains cell killing activity, demonstrating that engagement of NKp30 alone is sufficient to promote innate cell activation and cytotoxicity, although activity is enhanced by CD16A engagement. Furthermore, CTX-8573 maintains its cytotoxic activity in presence of soluble BCMA or BCMA ligands APRIL and BAFF. CTX-8573 does not induce innate cell activation, cytokine production, or killing in the absence of BCMA expressing target cells, supporting a wide therapeutic window. Additionally, unlike daratumumab, CTX-8573 does not induce NK-cell fratricide. In-vivo, CTX-8573 demonstrates anti-tumor efficacy in multiple humanized mouse models including killing of low BCMA expressing cell lines. In Cynomolgus monkeys, CTX-8573 displays standard biphasic pharmacokinetics with a 16 day β-phase half-life and has no evidence of systemic immune activation as measured by C-reactive protein levels. Lastly, NKp30 expression is maintained on bone marrow NK cells from MM patients including the presence of a significant NKp30+CD16a- subpopulation. Conclusion: CTX-8573 represents a novel class of bispecific antibodies that promote potent tumor cell killing by NK and γδ T-cells through engagement of the activating receptors NKp30 and CD16a. CTX-8573 demonstrates strong anti-tumor efficacy in vitro and in vivo, a wide therapeutic window with no evidence of systemic toxicity, and monoclonal-like pharmacokinetics and manufacturability. Together, these data highlight the potential of CTX-8573 as a novel treatment for MM either alone or as a complement to existing therapies. Disclosures Watkins-Yoon: Compass therapeutics LLC: Employment, Equity Ownership. Guzman:Compass therapeutics LLC: Employment, Equity Ownership. Oliphant:Compass therapeutics LLC: Employment, Equity Ownership. Haserlat:Compass therapeutics LLC: Employment, Equity Ownership. Leung:Compass therapeutics LLC: Employment, Equity Ownership. Chottin:University of Louisiana at Lafayette: Employment. Ophir:Compass therapeutics LLC: Employment, Equity Ownership. Vekeria:Compass therapeutics LLC: Employment, Equity Ownership. Nanjappa:Compass therapeutics LLC: Employment, Equity Ownership. Markrush:Compass therapeutics LLC: Employment, Equity Ownership. McConaughy:Compass therapeutics LLC: Employment, Equity Ownership. Wang:Compass therapeutics LLC: Employment, Equity Ownership. Schilling:Compass therapeutics LLC: Employment, Equity Ownership. Kim:Compass therapeutics LLC: Employment, Equity Ownership. Wu:Compass Therapeutics LLC: Employment, Equity Ownership. Liu:Compass therapeutics LLC: Employment, Equity Ownership. Rogers:University of Louisiana at Lafayette: Employment. Villinger:University of Louisiana at Lafayette: Employment. Gong:Compass therapeutics LLC: Employment, Equity Ownership. Hamilton:Compass therapeutics LLC: Employment, Equity Ownership. Bobrowicz:Compass therapeutics LLC: Employment, Equity Ownership. Schuetz:Compass therapeutics LLC: Employment, Equity Ownership. Schmidt:Compass therapeutics LLC: Employment, Equity Ownership. Draghi:Compass therapeutics LLC: Employment, Equity Ownership.

Pax transactivation domain-interacting protein (PTIP) is a ubiquitously expressed, nuclear protein that is part of a histone H3K4 methyltransferase complex and is essential for embryonic development. Methylation of H3K is an epigenetic mark found at many critical developmental regulatory genes in embryonic stem cells (ES) and, together with H3K27 methylation, constitutes a bivalent epigenetic signature. To address the function of PTIP in ES cells, we generated ES cell lines from a floxed ptip allele and deleted PTIP function with Cre recombinase. The ptip−/− ES cell lines exhibited a high degree of spontaneous differentiation to trophectoderm and a loss of pluripotency. Reduced levels of Oct4 expression and H3K4 methylation were observed. Upon differentiation, ptip−/− embryoid bodies showed reduced levels of marker gene expression for all three primary germ layers. These results suggest that the maintenance of H3K4 methylation is essential and requires PTIP function during the in vitro propagation of pluripotent ES cells.

Pax transactivation-domain interacting protein (PTIP) is a widely expressed nuclear protein that is essential for early embryonic development. PTIP was first identified on the basis of its interactions with the developmental regulator Pax2 but can also bind to other nuclear transcription factors. The Pax2 protein is essential for development of the renal epithelia and for regulating the response of mature collecting ducts to hyperosmotic stress. For determination of whether PTIP also functions in more differentiated cell types, the Cre-LoxP system was used to delete the ptip gene in the renal collecting ducts using Ksp-Cre driver mice. Collecting duct-specific ptip knockout mice were viable with little discernible phenotype under normal physiologic conditions. However, collecting duct-specific ptip mutants were unable to concentrate urine after the treatment of desamino-cis, D-arginine vasopressin, an antidiuretic hormone. Furthermore, aquaporin-2 (AQP2) expression in the inner medulla of the ptip knockout mice was decreased approximately 10-fold compared with that of wild-type littermates. Expression level of tonicity responsive enhancer binding protein, a transcription factor of AQP2, is not altered in the mutant mice, but its nuclear localization in the inner medulla is unresponsive after treatment with vasopressin agonists. This was due, at least in part, to decreased expression of the arginine vasopressin receptor 2 in ptip mutants. Furthermore, ptip null inner medullary collecting duct cells were sensitive to hyperosmolality in vitro. Thus, ptip is required for the urine concentration mechanism by modulating arginine vasopressin receptor 2 and AQP2 expression in the inner medulla. The data suggest an essential role for ptip in regulating urine concentration and in controlling survival of collecting duct epithelial cells in high osmolality.