Macrocyclic Carbon-Linked Pyrazoles As Novel Inhibitors of MCL-1.

Myeloid cell leukemia-1 (MCL-1) is a member of the antiapoptotic BCL-2 proteins family and a key regulator of mitochondrial homeostasis. Overexpression of MCL-1 is found in many cancer cells and contributes to tumor progression, which makes it an attractive therapeutic target. Pursuing our previous study of macrocyclic indoles for the inhibition of MCL-1, we report herein the impact of both pyrazole and indole isomerism on the potency and overall properties of this family of compounds. We demonstrated that the incorporation of a fluorine atom on the naphthalene moiety was a necessary step to improve cellular potency and that, combined with the introduction of various side chains on the pyrazole, it enhanced solubility significantly. This exploration culminated in the discovery of compounds (Ra)-10 and (Ra)-15, possessing remarkable cellular potency and properties.

Discovery of an Oral, Beyond-Rule-of-Five Mcl-1 Protein-Protein Interaction Modulator with the Potential of Treating Hematological Malignancies.

Avoidance of apoptosis is critical for the development and sustained growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family of proteins which is overexpressed in many cancers. Upregulation of Mcl-1 in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Therefore, pharmacological inhibition of Mcl-1 is regarded as an attractive approach to treating relapsed or refractory malignancies. Herein, we disclose the design, synthesis, optimization, and early preclinical evaluation of a potent and selective small-molecule inhibitor of Mcl-1. Our exploratory design tactics focused on structural modifications which improve the potency and physicochemical properties of the inhibitor while minimizing the risk of functional cardiotoxicity. Despite being in the "non-Lipinski" beyond-Rule-of-Five property space, the developed compound benefits from exquisite oral bioavailability in vivo and induces potent pharmacodynamic inhibition of Mcl-1 in a mouse xenograft model.

The methyltransferase domain of DNMT1 is an essential domain in acute myeloid leukemia independent of DNMT3A mutation.

Aberrant DNA methylation patterns are a prominent feature of cancer. Methylation of DNA is mediated by the DNA methyltransferase (DNMT) protein family, which regulates de novo (DNMT3A and DNMT3B) and maintenance (DNMT1) methylation. Mutations in DNMT3A are observed in approximately 22% of acute myeloid leukemia (AML). We hypothesized that DNMT1 or DNMT3B could function as a synthetic lethal therapeutic strategy for DNMT3A-mutant AML. CRISPR-Cas9 tiling screens were performed to identify functional domains within DNMT1/DNMT3B that exhibited greater dependencies in DNMT3A mutant versus wild-type cell lines. Although increased sensitivity to DNMT1 mutation was observed in some DNMT3A mutant cellular models tested, the subtlety of these results prevents us from basing any conclusions on a synthetic lethal relationship between DNMT1 and DNMT3A. Our data suggests that a therapeutic window for DNMT1 methyltransferase inhibition in DNMT3A-driven AML may exist, but validation in more biologically relevant models is required.

Discovery of JNJ-64264681: A Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.

BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL.

Activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCL) have unfavorable outcomes and chronic activation of CARD11-BCL10-MALT1 (CBM) signal amplification complexes that form due to polymerization of BCL10 subunits, which is affected by recurrent somatic mutations in ABC-DLBCLs. Herein, we show that BCL10 mutants fall into at least two functionally distinct classes: missense mutations of the BCL10 CARD domain and truncation of its C-terminal tail. Truncating mutations abrogated a motif through which MALT1 inhibits BCL10 polymerization, trapping MALT1 in its activated filament-bound state. CARD missense mutations enhanced BCL10 filament formation, forming glutamine network structures that stabilize BCL10 filaments. Mutant forms of BCL10 were less dependent on upstream CARD11 activation and thus manifested resistance to BTK inhibitors, whereas BCL10 truncating but not CARD mutants were hypersensitive to MALT1 inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL, and further precision can be achieved by selecting therapy based on specific biochemical effects of distinct mutation classes. SIGNIFICANCE: ABC-DLBCLs feature frequent mutations of signaling mediators that converge on the CBM complex. We use structure-function approaches to reveal that BCL10 mutations fall into two distinct biochemical classes. Both classes confer resistance to BTK inhibitors, whereas BCL10 truncations confer hyperresponsiveness to MALT1 inhibitors, providing a road map for precision therapies in ABC-DLBCLs. See related commentary by Phelan and Oellerich, p. 1844. This article is highlighted in the In This Issue feature, p. 1825.

Discovery and Pharmacological Characterization of JNJ-64619178, a Novel Small-Molecule Inhibitor of PRMT5 with Potent Antitumor Activity.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors.

Discovery of Highly Potent and Selective IRAK1 Degraders to Probe Scaffolding Functions of IRAK1 in ABC DLBCL.

MyD88 gene mutation has been identified as one of the most prevalent driver mutations in the activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL). The published literature suggests that interleukin-1 receptor-associated kinase 1 (IRAK1) is an essential gene for ABC DLBCL harboring MyD88 mutation. Importantly, the scaffolding function of IRAK1, rather than its kinase activity, is required for tumor cell survival. Herein, we present our design, synthesis, and biological evaluation of a novel series of potent and selective IRAK1 degraders. One of the most potent compounds, Degrader-3 (JNJ-1013), effectively degraded cellular IRAK1 protein with a DC50 of 3 nM in HBL-1 cells. Furthermore, JNJ-1013 potently inhibited IRAK1 downstream signaling pathways and demonstrated strong anti-proliferative effects in ABC DLBCL cells with MyD88 mutation. This work suggests that IRAK1 degraders have the potential for treating cancers that are dependent on the IRAK1 scaffolding function.

Assessing IRAK4 Functions in ABC DLBCL by IRAK4 Kinase Inhibition and Protein Degradation.

The interleukin-1 receptor-activated kinase 4 (IRAK4) belongs to the IRAK family of serine/threonine kinases and plays a central role in the innate immune response. However, the function of IRAK4 in tumor growth and progression remains elusive. Here we sought to determine the enzymatic and scaffolding functions of IRAK4 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). We chose a highly selective IRAK4 kinase inhibitor to probe the biological effects of kinase inhibition and developed a series of IRAK4 degraders to evaluate the effects of protein degradation in ABC DLBCL cells. Interestingly, the results demonstrated that neither IRAK4 kinase inhibition nor protein degradation led to cell death or growth inhibition, suggesting a redundant role for IRAK4 in ABC DLBCL cell survival. IRAK4 degraders characterized in this study provide useful tools for understanding IRAK4 protein scaffolding function, which was previously unachievable using pharmacological perturbation.

Discovery and optimization of a series of small-molecule allosteric inhibitors of MALT1 protease.

We describe a series of potent and highly selective small-molecule MALT1 inhibitors, optimized from a High-Throughput Screening hit. Advanced analogues such as compound 40 show high potency (IC50: 0.01 µM) in a biochemical assay measuring MALT1 enzymatic activity, as well as in cellular assays: Jurkat T cell activation (0.05 µM) and IL6/10 secretion (IC50: 0.10/0.06 µM) in the TMD8 B-cell lymphoma line. Compound 40 also inhibited cleavage of the MALT1 substrate RelB (IC50: 0.10 µM). Mechanistic enzymology results suggest that these compounds bind to the known allosteric site of the protease.

Influence of the bispecific antibody IgG subclass on T cell redirection.

T cell redirection mediated by bispecific antibodies (BsAbs) is a promising cancer therapy. Dual antigen binding is necessary for potent T cell redirection and is influenced by the structural characteristics of a BsAb, which are dependent on its IgG subclass. In this study, model BsAbs targeting CD19xCD3 were generated in variants of IgG1, IgG2, and IgG4 carrying Fc mutations that reduce FcγR interaction, and two chimeric IgG subclasses termed IgG1:2 and IgG4:2, in which the IgG1- or IgG4-F(ab)2 are grafted on an IgG2 Fc. Molecules containing an IgG2 or IgG4-F(ab)2 domain were confirmed to be the most structurally compact molecules. All BsAbs were shown to bind both of their target proteins (and corresponding cells) equally well. However, CD19xCD3 IgG2 did not bind both antigens simultaneously as measured by the absence of cellular clustering of T cells with target cells. This translated to a reduced potency of IgG2 BsAbs in T-cell redirection assays. The activity of IgG2 BsAbs was fully restored in the chimeric subclasses IgG4:2 and IgG1:2. This confirmed the major contribution of the F(ab)2 region to the BsAb's functional activity and demonstrated that function of BsAbs can be modulated by engineering molecules combining different Fc and F(ab)2 domains. Abbreviations: ADCC: Antibody-dependent cellular cytotoxicity; AlphaScreenTM: Amplified Luminescent Proximity Homogeneous Assay Screening; ANOVA: Analysis of variance; BiTE: bispecific T-cell engager; BSA: bovine serum albumin; BsAb: bispecific antibody; cFAE: controlled Fab-arm exchange; CDC: complement-dependent cellular cytotoxicity; CIEX: cation-exchange; CIR: chimeric immune receptor; DPBS: Dulbecco's phosphate-buffered saline; EC50 value: effective concentration to reach half-maximum effect; EGFR: epidermal growth factor receptor; EI: expansion index (RAt=x/RAt=0); FACS: fluorescence-activated cell sorting; FVD: fixable viability dye; HI-HPLC: hydrophobic interaction HPLC; HI-FBS: heat-inactivated fetal bovine serum; HPLC: high-pressure liquid chromatography; IC50 value: effective concentration to reach half-maximum inhibition; IQ: Inhibition Quotient; IS: immunological synapse; MES: 2-(N-morpholino)ethanesulfonic acid; R-PE: recombinant phycoerythrin; RA: red area in µm2/well; RD: receptor density; RFP: red fluorescent protein; Rg: radius of gyration; RSV: respiratory syncytial virus; SAXS: small-angle x-ray scattering; scFv: single-chain variable fragment; SD: standard deviation; SPR: surface plasmon resonance; WT: wild-type.

Specific covalent inhibition of MALT1 paracaspase suppresses B cell lymphoma growth.

The paracaspase MALT1 plays an essential role in activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) downstream of B cell and TLR pathway genes mutated in these tumors. Although MALT1 is considered a compelling therapeutic target, the development of tractable and specific MALT1 protease inhibitors has thus far been elusive. Here, we developed a target engagement assay that provides a quantitative readout for specific MALT1-inhibitory effects in living cells. This enabled a structure-guided medicinal chemistry effort culminating in the discovery of pharmacologically tractable, irreversible substrate-mimetic compounds that bind the MALT1 active site. We confirmed that MALT1 targeting with compound 3 is effective at suppressing ABC DLBCL cells in vitro and in vivo. We show that a reduction in serum IL-10 levels exquisitely correlates with the drug pharmacokinetics and degree of MALT1 inhibition in vitro and in vivo and could constitute a useful pharmacodynamic biomarker to evaluate these compounds in clinical trials. Compound 3 revealed insights into the biology of MALT1 in ABC DLBCL, such as the role of MALT1 in driving JAK/STAT signaling and suppressing the type I IFN response and MHC class II expression, suggesting that MALT1 inhibition could prime lymphomas for immune recognition by cytotoxic immune cells.

Discovery of 1-(1H-Pyrazolo[4,3-c]pyridin-6-yl)urea Inhibitors of Extracellular Signal-Regulated Kinase (ERK) for the Treatment of Cancers.

The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is activated in more than 30% of human cancers. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the ERK signal pathway. Acquired resistance to these agents has led to greater interest in ERK, a downstream target of the MAPK pathway. De novo design efforts of a novel scaffold derived from SCH772984 by employing hydrogen bond interactions specific for ERK in the binding pocket identified 1-(1H-pyrazolo[4,3-c]pyridin-6-yl)ureas as a viable lead series. Sequential SAR studies led to the identification of highly potent and selective ERK inhibitors with low molecular weight and high LE. Compound 21 exhibited potent target engagement and strong tumor regression in the BRAF(V600E) xenograft model.

PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes.

Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors.

The high frequency of activating RAS or BRAF mutations in cancer provides strong rationale for targeting the mitogen-activated protein kinase (MAPK) pathway. Selective BRAF and MAP-ERK kinase (MEK) inhibitors have shown clinical efficacy in patients with melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the extracellular signal-regulated kinase (ERK) signaling pathway. Here, we describe the identification and characterization of SCH772984, a novel and selective inhibitor of ERK1/2 that displays behaviors of both type I and type II kinase inhibitors. SCH772984 has nanomolar cellular potency in tumor cells with mutations in BRAF, NRAS, or KRAS and induces tumor regressions in xenograft models at tolerated doses. Importantly, SCH772984 effectively inhibited MAPK signaling and cell proliferation in BRAF or MEK inhibitor-resistant models as well as in tumor cells resistant to concurrent treatment with BRAF and MEK inhibitors. These data support the clinical development of ERK inhibitors for tumors refractory to MAPK inhibitors.

Gene signatures derived from a c-MET-driven liver cancer mouse model predict survival of patients with hepatocellular carcinoma.

Biomarkers derived from gene expression profiling data may have a high false-positive rate and must be rigorously validated using independent clinical data sets, which are not always available. Although animal model systems could provide alternative data sets to formulate hypotheses and limit the number of signatures to be tested in clinical samples, the predictive power of such an approach is not yet proven. The present study aims to analyze the molecular signatures of liver cancer in a c-MET-transgenic mouse model and investigate its prognostic relevance to human hepatocellular carcinoma (HCC). Tissue samples were obtained from tumor (TU), adjacent non-tumor (AN) and distant normal (DN) liver in Tet-operator regulated (TRE) human c-MET transgenic mice (n = 21) as well as from a Chinese cohort of 272 HBV- and 9 HCV-associated HCC patients. Whole genome microarray expression profiling was conducted in Affymetrix gene expression chips, and prognostic significances of gene expression signatures were evaluated across the two species. Our data revealed parallels between mouse and human liver tumors, including down-regulation of metabolic pathways and up-regulation of cell cycle processes. The mouse tumors were most similar to a subset of patient samples characterized by activation of the Wnt pathway, but distinctive in the p53 pathway signals. Of potential clinical utility, we identified a set of genes that were down regulated in both mouse tumors and human HCC having significant predictive power on overall and disease-free survival, which were highly enriched for metabolic functions. In conclusions, this study provides evidence that a disease model can serve as a possible platform for generating hypotheses to be tested in human tissues and highlights an efficient method for generating biomarker signatures before extensive clinical trials have been initiated.

Predictive genes in adjacent normal tissue are preferentially altered by sCNV during tumorigenesis in liver cancer and may rate limiting.

BACKGROUND: In hepatocellular carcinoma (HCC) genes predictive of survival have been found in both adjacent normal (AN) and tumor (TU) tissues. The relationships between these two sets of predictive genes and the general process of tumorigenesis and disease progression remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we have investigated HCC tumorigenesis by comparing gene expression, DNA copy number variation and survival using ∼250 AN and TU samples representing, respectively, the pre-cancer state, and the result of tumorigenesis. Genes that participate in tumorigenesis were defined using a gene-gene correlation meta-analysis procedure that compared AN versus TU tissues. Genes predictive of survival in AN (AN-survival genes) were found to be enriched in the differential gene-gene correlation gene set indicating that they directly participate in the process of tumorigenesis. Additionally the AN-survival genes were mostly not predictive after tumorigenesis in TU tissue and this transition was associated with and could largely be explained by the effect of somatic DNA copy number variation (sCNV) in cis and in trans. The data was consistent with the variance of AN-survival genes being rate-limiting steps in tumorigenesis and this was confirmed using a treatment that promotes HCC tumorigenesis that selectively altered AN-survival genes and genes differentially correlated between AN and TU. CONCLUSIONS/SIGNIFICANCE: This suggests that the process of tumor evolution involves rate-limiting steps related to the background from which the tumor evolved where these were frequently predictive of clinical outcome. Additionally treatments that alter the likelihood of tumorigenesis occurring may act by altering AN-survival genes, suggesting that the process can be manipulated. Further sCNV explains a substantial fraction of tumor specific expression and may therefore be a causal driver of tumor evolution in HCC and perhaps many solid tumor types.

DLK1-DIO3 genomic imprinted microRNA cluster at 14q32.2 defines a stemlike subtype of hepatocellular carcinoma associated with poor survival.

Hepatocellular carcinoma (HCC) is a heterogeneous and highly aggressive malignancy, for which there are no effective cures. Identification of a malignant stemlike subtype of HCC may offer patients with a dismal prognosis a potential targeted therapy using c-MET and Wnt pathway inhibitors. MicroRNAs (miRNAs) show promise as diagnostic and prognostic tools for cancer detection and stratification. Using a TRE-c-Met-driven transgenic HCC mouse model, we identified a cluster of 23 miRNAs that is encoded within the Dlk1-Gtl2 imprinted region on chromosome 12qF1 overexpressed in all of the isolated liver tumors. Interestingly, this region is conserved among mammalian species and maps to the human DLK1-DIO3 region on chromosome 14q32.2. We thus examined the expression of the DLK1-DIO3 miRNA cluster in a cohort of 97 hepatitis B virus-associated HCC patients and identified a subgroup (n = 18) of patients showing strong coordinate overexpression of miRNAs in this cluster but not in other cancer types (breast, lung, kidney, stomach, and colon) that were tested. Expression levels of imprinted gene transcripts from neighboring loci in this 14q32.2 region and from a subset of other imprinted sites were concomitantly elevated in human HCC. Interestingly, overexpression of the DLK1-DIO3 miRNA cluster was positively correlated with HCC stem cell markers (CD133, CD90, EpCAM, Nestin) and associated with a high level of serum α-fetoprotein, a conventional biomarker for liver cancer, and poor survival rate in HCC patients. In conclusion, our findings suggest that coordinate up-regulation of the DLK1-DIO3 miRNA cluster at 14q32.2 may define a novel molecular (stem cell-like) subtype of HCC associated with poor prognosis.

Degeneration of the mouse retina upon dysregulated activity of serum response factor.

PURPOSE: Our aim was to generate and phenotypically characterize a transgenic mouse line expressing a constitutively active variant of the transcription regulatory protein serum response factor (SRF), namely the SRF-VP16 protein. This new mouse strain has been registered under the designation Gt(ROSA)26Sor(tm1(SRF-VP16)Antu). We found phenotypic changes upon ectopic expression of SRF-VP16, especially in the mouse retina. METHODS: Using homologous recombination, we integrated an SRF-VP16 conditional (i.e., "flox-STOP" repressed) expression transgene into the Rosa26 locus of murine embryonic stem (ES) cells. These engineered ES cells were used to derive the Gt(ROSA)26Sor(tm1(SRF-VP16)Antu) mouse strain. Semiquantitative real-time PCR was used to determine expression of the SRF-VP16 transgene at the mRNA level, both in young (P20 and P30) and adult (six months old) Gt(ROSA)26Sor(tm1(SRF-VP16)Antu) mice. We also investigated the transcript levels of endogenous Srf and several SRF target genes. Retinal function was tested by electroretinography in both young and adult mice. Morphological abnormalities could be visualized by hematoxylin and eosin staining of sectioned, paraffin-embedded eye tissue samples. Scanning-laser ophthalmoscopy was used to investigate retinal vascularization and degeneration in adult mice. RESULTS: We show that the SRF-VP16 mRNA is expressed to a low but significant degree in the retinas of young and adult animals of the Gt(ROSA)26Sor(tm1(SRF-VP16)Antu) mouse strain, even in the absence of Cre-mediated deletion of the "flox-STOP" cassette. In the retinas of these transgenic mice, endogenous Srf displays elevated transcript levels. Ectopic retinal expression of constitutively active SRF-VP16 is correlated with the malfunction of retinal neurons in both heterozygous and homozygous animals of both age groups (P20 and adult). Additionally, mislamination of retinal cell layers and cellular rosette formations are found in retinas of both heterozygous and homozygous animals of young age. In homozygous individuals, however, the cellular rosettes are more widespread over the fundus. At adult age, retinas both from animals that are heterozygous and homozygous for the floxSTOP/SRF-VP16 transgene display severe degeneration, mainly of the photoreceptor cell layer. Wild-type age-matched littermates, however, do not show any degeneration. The severity of the observed effects correlates with dosage of the transgene. CONCLUSIONS: This is the first report suggesting an influence of the transcription factor SRF on the development and function of the murine retina. Ectopic SRF-VP16 mRNA expression in the retinas of young animals is correlated with photoreceptor layer mislamination and impaired retinal function. At an advanced age of six months, degenerative processes are detected in SRF-VP16 transgenic retinas accompanied by impaired retinal function. The Gt(ROSA)26Sor(tm1(SRF-VP16)Antu) mouse strain represents a genetic SRF gain-of-function mouse model that will complement the current SRF loss-of-function models. It promises to provide new insight into the hitherto poorly defined role of SRF in retinal development and function, including potential contributions to ophthalmologic disorders. Furthermore, using conditional Cre-mediated activation of SRF-VP16, the described mouse strain will enable assessment of the impact of dysregulated SRF activity on the physiologic functions of various other organs.

Identification of invasion specific splice variants of the cytoskeletal protein Mena present in mammary tumor cells during invasion in vivo.

We have studied the gene expression pattern of invasive primary mammary tumor cells using a unique in vivo invasion assay that isolates the invasive tumor cells by chemotaxis. One of the genes upregulated in the invasive tumor cells is Mena, an actin binding protein involved in the regulation of cell motility. There are multiple known splice variants of Mena accounted for by four alternatively included exons, +, ++, +++ and 11a. Using the in vivo invasion assay in rats and mice with mammary tumors we observed that two isoforms of Mena, ++ and +++, are upregulated in the invasive tumor cells and one isoform, 11a, is downregulated. The Mena isoform switching pattern described here may provide a new biomarker for the presence of metastatic cancer cells and for prognosis.

A Mena invasion isoform potentiates EGF-induced carcinoma cell invasion and metastasis.

The spread of cancer during metastatic disease requires that tumor cells subvert normal regulatory networks governing cell motility to invade surrounding tissues and migrate toward blood and lymphatic vessels. Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) proteins regulate cell motility by controlling the geometry of assembling actin networks. Mena, an Ena/VASP protein, is upregulated in the invasive subpopulation of breast cancer cells. In addition, Mena is alternately spliced to produce an invasion isoform, Mena(INV). Here we show that Mena and Mena(INV) promote carcinoma cell motility and invasiveness in vivo and in vitro, and increase lung metastasis. Mena and Mena(INV) potentiate epidermal growth factor (EGF)-induced membrane protrusion and increase the matrix degradation activity of tumor cells. Interestingly, Mena(INV) is significantly more effective than Mena in driving metastases and sensitizing cells to EGF-dependent invasion and protrusion. Upregulation of Mena(INV) could therefore enable tumor cells to invade in response to otherwise benign EGF stimulus levels.