Structural maintenance of chromosome-1A (SMC1A) is overexpressed in various malignancies including triple-negative breast cancer (TNBC). As a core component of the cohesin complex, SMC1A was initially recognized for its involvement in chromosomal cohesion and DNA-repair pathways. However, recent studies have unveiled its pivotal role in epithelial-mesenchymal transition (EMT), metastasis, and chemo- and radio-resistance in cancer cells. In hepatocellular carcinoma, aberrant phosphorylation of SMC1A has been associated with enhanced cell proliferation and migration. Despite these insights, the precise role of SMC1A phosphorylation in breast cancer remains largely unexplored. This study represents the first investigation to test the phosphorylation status and subcellular localization of SMC1A (p-SMC1A) in breast cancer and normal breast tissues. Immunohistochemical (IHC) staining was conducted using previously validated phospho-SMC1A antibodies on a histological section and tissue microarray (TMA) comprising samples from primary, invasive, and metastatic breast cancer and normal breast tissues. Our results revealed that p-SMC1A staining intensity was lower in normal breast tissues compared to invasive or metastatic breast cancer tissues (p < 0.001). Approximately 40% of breast cancer tissue exhibited cytoplasmic/membranous localization of p-SMC1A, whereas nuclear expression was observed in normal breast tissues. Moreover, elevated phosphorylation levels were significantly associated with higher tumor grade and metastasis.

Figure S1. Increased TLR9 expression in induced glioma spheres. Figure S2. TLR9 and STAT3 form a feed-forward loop in GSCs. Figure S3. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits tumor growth. Figure S4. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits GSCs. Figure S5. Targeting Brain tumors systemically with CpG-siRNA reaches the tumor site.

Supplementary Figure 1 from STAT3 Inhibition Is a Therapeutic Strategy for ABC-like Diffuse Large B-Cell Lymphoma

Supplementary Figures 1-6 from Combining Histone Deacetylase Inhibitor Vorinostat with Aurora Kinase Inhibitors Enhances Lymphoma Cell Killing with Repression of c-Myc, hTERT, and microRNA Levels

Supplementary Table 1 from Combining Histone Deacetylase Inhibitor Vorinostat with Aurora Kinase Inhibitors Enhances Lymphoma Cell Killing with Repression of c-Myc, hTERT, and microRNA Levels

Supplementary Figure Legends 1-3 from STAT3 Inhibition Is a Therapeutic Strategy for ABC-like Diffuse Large B-Cell Lymphoma

PDF file - 5546K, Supplementary Fig.2: STAT3 activation is associated with upregulated CSC marker expression.

Supplementary Figure 2 from Targeting Stat3 in the Myeloid Compartment Drastically Improves the In vivo Antitumor Functions of Adoptively Transferred T Cells

PDF file - 1025K, Supplementary Fig. 1: Elevated STAT3 activity results in enhanced tumor growth.

PDF file - 2401K, Supplementary Fig. 3: Characterization of recombinant IL-6RFP.

Supplementary Figure 1 from Targeting Stat3 in the Myeloid Compartment Drastically Improves the In vivo Antitumor Functions of Adoptively Transferred T Cells

Supplementary Figure Legends for Table 1 and Figures 1-6 from Combining Histone Deacetylase Inhibitor Vorinostat with Aurora Kinase Inhibitors Enhances Lymphoma Cell Killing with Repression of c-Myc, hTERT, and microRNA Levels

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Cutaneous T cell lymphomas (CTCLs) are a heterogeneous group of debilitating, incurable malignancies. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes, accounting for ~65% of CTCL cases. Patients with advanced disease have a poor prognosis and low median survival rates of four years. CTCLs develop from malignant skin-homing CD4+ T cells that spread to lymph nodes, blood, bone marrow and viscera in advanced stages. Current treatments options for refractory or advanced CTCL, including chemotherapeutic and biological approaches, rarely lead to durable responses. The exact molecular mechanisms of CTCL pathology remain unclear despite numerous genomic and gene expression profile studies. However, apoptosis resistance is thought to play a major role in the accumulation of malignant T cells. Here we show that NT1721, a synthetic epidithiodiketopiperazine based on a natural product, reduced cell viability at nanomolar concentrations in CTCL cell lines, while largely sparing normal CD4+ cells. Treatment of CTCL cells with NT1721 reduced proliferation and potently induced apoptosis. NT1721 mediated the downregulation of GLI1 transcription factor, which was associated with decreased STAT3 activation and the reduced expression of downstream antiapoptotic proteins (BCL2 and BCL-xL). Importantly, NT1721, which is orally available, reduced tumor growth in two CTCL mouse models significantly better than two clinically used drugs (romidepsin, gemcitabine). Moreover, a combination of NT1721 with gemcitabine reduced the tumor growth significantly better than the single drugs. Taken together, these results suggest that NT1721 may be a promising new agent for the treatment of CTCLs.

Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor-risk FLT3-ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8-chloro-adenosine (8-Cl-Ado) is a ribose-containing, RNA-directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8-Cl-Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8-Cl-Ado in AML. 8-Cl-Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8-Cl-Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3-ITD mutation. FLT3-ITD is associated with high expression of miR-155. Furthermore, we demonstrate that 8-Cl-Ado inhibits miR-155 expression levels accompanied by induction of DNA-damage and suppression of cell proliferation, through regulation of miR-155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3-ITD (+) MV4-11 AML cells with 8-Cl-Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3-ITD AML patients.

The multifunctional human DNA2 (hDNA2) nuclease/helicase is required to process DNA ends for homology-directed recombination repair (HDR) and to counteract replication stress. To participate in these processes, hDNA2 must localize to the nucleus and be recruited to the replication or repair sites. However, because hDNA2 lacks the nuclear localization signal that is found in its yeast homolog, it is unclear how its migration into the nucleus is regulated during replication or in response to DNA damage. Here, we report that the E3 ligase TRAF6 binds to and mediates the K63-linked polyubiquitination of hDNA2, increasing the stability of hDNA2 and promoting its nuclear localization. Inhibiting TRAF6-mediated polyubiquitination abolishes the nuclear localization of hDNA2, consequently impairing DNA end resection and HDR. Thus, the current study reveals a mechanism for the regulation of hDNA2 localization and establishes that TRAF6-mediated hDNA2 ubiquitination activates DNA repair pathways to maintain nuclear genome integrity.

Background Pancreatic cancer is one of the most lethal malignancies due to frequent late diagnosis, aggressive tumor growth and metastasis formation. Continuously raising incidence rates of pancreatic cancer and a lack of significant improvement in survival rates over the past 30 years highlight the need for new therapeutic agents. Thus, new therapeutic agents and strategies are urgently needed to improve the outcome for patients with pancreatic cancer. Here, we evaluated the anti-tumor activity of a new natural product-based epidithiodiketopiperazine, NT1721, against pancreatic cancer. Methods We characterized the anticancer efficacy of NT1721 in multiple pancreatic cancer cell lines in vitro and in two orthotopic models. We also compared the effects of NT1721 to clinically used hedgehog inhibitors and the standard-of-care drug, gemcitabine. The effect of NT1721 on hedgehog/GLI signaling was assessed by determining the expression of GLI and GLI target genes both in vitro and in vivo . Results NT1721 displayed IC 50 values in the submicromolar range in multiple pancreatic cancer cell lines, while largely sparing normal pancreatic epithelial cells. NT1721 attenuated hedgehog/GLI signaling through downregulation of GLI1/2 transcription factors and their downstream target genes, which reduced cell proliferation and invasion in vitro and significantly decreased tumor growth and liver metastasis in two preclinical orthotopic mouse models of pancreatic cancer. Importantly, treatment with NT1721 significantly improved survival times of mice with pancreatic cancer compared to the standard-of-care drug, gemcitabine. Conclusions Favorable therapeutics properties, i.e. 10-fold lower IC 50 values than clinically used hedgehog inhibitors (vismodegib, erismodegib), a 90% reduction in liver metastasis and significantly better survival times compared to the standard-of-care drug, gemcitabine, provide a rational for testing NT1721 in the clinic either as a single agent or possibly in combination with gemcitabine or other therapeutic agents in PDAC patients overexpressing GLI1/2. This could potentially result in promising new treatment options for patients suffering from this devastating disease.

AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362]. As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values <1 at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90)]. In an orthotopic mouse model where FLT3-ITD-positive MV4-11 cells were xenografted, mice treated with 75 mg 8-Cl-Ado/kg/day plus quizartinib (0.5 mg/kg/day) survived significantly longer than mice treated with the individual agents or vehicle alone (median survival 54.2 days vs. 27.3 days for controls, p=0.016).

Prostate cancer is one of the most commonly diagnosed cancers and a pressing health challenge in men worldwide. Radiation therapy (RT) is widely considered a standard therapy for advanced as well as localized prostate cancer. Although this primary therapy is associated with high cancer control rates, up to one‐third of patients undergoing radiation therapy becomes radio‐resistant and/or has tumor‐relapse/recurrence. Therefore, focus on new molecular targets and pathways is essential to develop novel radio‐sensitizing agents for the effective and safe treatment of prostate cancer. Here, we describe functional studies that were performed to investigate the role of structural maintenance of chromosome‐1 (SMC1A) in radioresistance of metastatic prostate cancer cells. Short hairpin RNA (shRNA) was used to suppress SMC1A in metastatic castration‐resistant prostate cancer cells, DU145 and PC3. Clonogenic survival assays, Western blot, RT‐PCR, and γ‐H2AX staining were used to assess the effect of SMC1A knockdown on radiation sensitivity of these prostate cancer cells. We demonstrate that SMC1A is overexpressed in human prostate tumors compared to the normal adjacent tissue. SMC1A knockdown limits the clonogenic potential, epithelial‐mesenchymal transition (EMT), and cancer stem‐like cell (CSC) properties of DU145 and PC3 cells and enhanced efficacy of RT in these cells. Targeted inhibition of SMC1A not only plays a critical role in overcoming radio‐resistance in prostate cancer cells, but also suppresses self‐renewal and the tumor‐propagating potential of x‐irradiated cancer cells. We propose that SMC1A could be a potential molecular target for the development of novel radio‐sensitizing therapeutic agents for management of radio‐resistant metastatic prostate cancer.