Pharmacodynamic effects of the PARP inhibitor talazoparib (MDV3800, BMN 673) in patients with BRCA-mutated advanced solid tumors.

PURPOSE: Talazoparib is an inhibitor of the poly (ADP-ribose) polymerase (PARP) family of enzymes and is FDA-approved for patients with (suspected) deleterious germline BRCA1/2-mutated, HER2­negative, locally advanced or metastatic breast cancer. Because knowledge of the pharmacodynamic (PD) effects of talazoparib in patients has been limited to studies of PARP enzymatic activity (PARylation) in peripheral blood mononuclear cells, we developed a study to assess tumoral PD response to talazoparib treatment (NCT01989546). METHODS: We administered single-agent talazoparib (1 mg/day) orally in 28-day cycles to adult patients with advanced solid tumors harboring (suspected) deleterious BRCA1 or BRCA2 mutations. The primary objective was to examine the PD effects of talazoparib; the secondary objective was to determine overall response rate (ORR). Tumor biopsies were mandatory at baseline and post-treatment on day 8 (optional at disease progression). Biopsies were analyzed for PARylation, DNA damage response (γH2AX), and epithelial‒mesenchymal transition. RESULTS: Nine patients enrolled in this trial. Four of six patients (67%) evaluable for the primary PD endpoint exhibited a nuclear γH2AX response on day 8 of treatment, and five of six (83%) also exhibited strong suppression of PARylation. A transition towards a more mesenchymal phenotype was seen in 4 of 6 carcinoma patients, but this biological change did not affect γH2AX or PAR responses. The ORR was 55% with the five partial responses lasting a median of six cycles. CONCLUSION: Intra-tumoral DNA damage response and inhibition of PARP enzymatic activity were confirmed in patients with advanced solid tumors harboring BRCA1/2 mutations after 8 days of talazoparib treatment.

Fine-needle aspiration cytology of epithelioid malignant peripheral nerve sheath tumor: A case report and review of the literature.

The epithelioid variant of malignant peripheral nerve sheath tumor (eMPNST) is an extremely rare soft tissue neoplasm comprising less than 5% of all MPNSTs. It is distinguished cytomorphologically from a conventional MPNST by the presence of polymorphous round epithelioid cells arranged in loose clusters with or without spindled tumor cells. These features pose a diagnostic challenge because the differential diagnosis involves a variety of mesenchymal and non-mesenchymal tumors including epithelioid sarcoma, sclerosing epithelioid fibrosarcoma, malignant rhabdoid tumor, chordoma, metastatic carcinomas, and melanoma. Thus, it may become imperative to perform immunochemical stains on cell blocks of FNA aspirates to arrive at definitive diagnosis. Reports describing the cytologic features of eMPNST are rare. Herein, we report a case of eMPNST with focus on cytomorphologic and cytoimmunochemical features and differential diagnosis.

TIMP-1 Inhibits Apoptosis in Lung Adenocarcinoma Cells via Interaction with Bcl-2.

Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target.

Biphenotypic plasma cell myeloma: two cases of plasma cell neoplasm with a coexpression of kappa and lambda light chains.

UNLABELLED: Plasma cell neoplasm (PCM) is a medullary and extra medullary proliferation of clonal plasma cells that occurs due to accidental translocation of proto-oncogenes into immunoglobulin (Ig) gene loci. While the majority of plasma cell neoplasms are monoclonal, up to 2% of the PCMs [1] considered being biclonal based on electrophoretic analysis, characterized by secretion of paraprotein with two distinct heavy chains or light chains are possible and present unique diagnostic challenges. METHODS: Traditionally protein electrophoresis has been used to diagnose, characterize, and monitor progression of plasma cell neoplasm. To characterize neoplastic plasma cells, in our institution, other ancillary studies, including in situ hybridization, flow cytometric analyses of plasma cell surface markers and cytoplasmic immunoglobulins with DNA ploidy, are also utilized routinely. RESULTS: We present two cases of plasma cell myeloma in which the neoplastic plasma cells shows production of cytoplasmic kappa and lambda light chain, with secretion of free lambda light chain only. Co-expression of kappa and lambda light chain by the same neoplastic plasma cells is a rare but reported phenomenon. CONCLUSIONS: Our study indicates that serum electrophoresis alone could mischaracterize biphenotypic myeloma as monotypic plasma cell myelomas in the absence of additional testing methods.

Diffuse large B-cell lymphoma of the colon--a rare presentation.

Patients with diffuse large B-cell lymphoma (DLBCL) can present with nodal or extra-nodal disease. The most common extra-nodal site is the gastrointestinal tract, including the stomach or illeo-cecal region. Primary colonic diffuse large B-cell lymphoma (DLBCL) is uncommon and presents a unique diagnostic and therapeutic challenge. We present a case of a middle-aged man who presented with abdominal pain and pathological weight loss and underwent hemi-colectomy for suspected adenocarcinoma. Final pathology revealed DLBCL, completely changing the prognosis and subsequent management. We will discuss some of the aspects of this rare presentation and shed light on therapeutic strategies currently available.

Endothelin-1 upregulation mediates aging-related cardiac fibrosis.

Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological conditions, such as hypertension and diabetes. Aging is an independent risk factor for myocardial fibrosis. We hypothesized that ET-1 upregulation may be a basis of enhanced collagen synthesis in the senescent fibroblasts resulting in cardiac fibrosis with aging. To examine this hypothesis, we cultured mouse cardiac fibroblasts to passage-30 (P30). ß-Galactosidase activity and several other aging markers were markedly increased in P30 (vs. P3) fibroblasts, indicating that these cells were indeed undergoing senescence. Importantly, ET-1 expression was markedly upregulated in P30 (vs. P3) fibroblasts. Of note, estrogen receptor-α (ER-α), an important negative regulator of ET-1, was downregulated in P30 fibroblasts. We also studied aged (130-weeks old, female) mice hearts, and observed that ET-1 was upregulated and ER-α was downregulated in these hearts (vs. 6-week old mice hearts, female). Similar observations were made in the fibroblasts isolated from aged mice hearts. ET-1 upregulation with aging was also seen in ≈70-year old (vs. ≈30-year old) human heart sections. In concert with ET-1 upregulation, the expression of fibronectin and collagens was found to be markedly increased in P30 cardiac fibroblasts in culture, fibroblasts isolated from the aged mice hearts, and in aged human hearts. Interestingly, inhibition of ET-1 in the senescent P30 fibroblasts by 2 different strategies (the use of siRNA and the use of endothelin converting enzyme inhibitors) markedly suppressed expression of fibrosis signals. Further, treatment with synthetic ET-1 enhanced fibronectin and collagen expression in P3 cardiac fibroblasts. These observations in mice and human hearts suggest that aging-related cardiac fibrosis is, at least partially, dependent on the upregulation of ET-1.

Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments.

All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells.

Chlamydia trachomatis Tarp cooperates with the Arp2/3 complex to increase the rate of actin polymerization.

Actin polymerization is required for Chlamydia trachomatis entry into nonphagocytic host cells. Host and chlamydial actin nucleators are essential for internalization of chlamydiae by eukaryotic cells. The host cell Arp2/3 complex and the chlamydial translocated actin recruiting phosphoprotein (Tarp) are both required for entry. Tarp and the Arp2/3 complex exhibit unique actin polymerization kinetics individually, but the molecular details of how these two actin nucleators cooperate to promote bacterial entry is not understood. In this study we provide biochemical evidence that the two actin nucleators act synergistically by co-opting the unique attributes of each to enhance the dynamics of actin filament formation. This process is independent of Tarp phosphorylation. We further demonstrate that Tarp colocalization with actin filaments is independent of the Tarp phosphorylation domain. The results are consistent with a model in which chlamydial and host cell actin nucleators cooperate to increase the rate of actin filament formation.

Identification of components of the host type IA phosphoinositide 3-kinase pathway that promote internalization of Listeria monocytogenes.

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria promotes its internalization into some human cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. The interaction of InlB with the Met receptor stimulates host signaling pathways that promote cell surface changes driving bacterial uptake. One human signaling protein that plays a critical role in Listeria entry is type IA phosphoinositide 3-kinase (PI 3-kinase). The molecular mechanism by which PI 3-kinase promotes bacterial internalization is not understood. Here we perform an RNA interference (RNAi)-based screen to identify components of the type IA PI 3-kinase pathway that control the entry of Listeria into the human cell line HeLa. The 64 genes targeted encode known upstream regulators or downstream effectors of type IA PI 3-kinase. The results of this screen indicate that at least 9 members of the PI 3-kinase pathway play important roles in Listeria uptake. These 9 human proteins include a Rab5 GTPase, several regulators of Arf or Rac1 GTPases, and the serine/threonine kinases phosphoinositide-dependent kinase 1 (PDK1), mammalian target of rapamycin (mTor), and protein kinase C-ζ. These findings represent a key first step toward understanding the mechanism by which type IA PI 3-kinase controls bacterial internalization.