Search

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action studies revealed the formation of a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts PRMT5-RIOK1 complexes, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive inhibitor that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

FK506-binding protein 35, FKBP35, has been implicated as an essential malarial enzyme. Rapamycin and FK506 exhibit antiplasmodium activity in cultured parasites. However, due to the highly conserved nature of the binding pockets of FKBPs and the immunosuppressive properties of these drugs, there is a need for compounds that selectively inhibit FKBP35 and lack the undesired side effects. In contrast to human FKBPs, FKBP35 contains a cysteine, C106, adjacent to the rapamycin binding pocket, providing an opportunity to develop targeted covalent inhibitors of Plasmodium FKBP35. Here, we synthesize inhibitors of FKBP35, show that they directly bind FKBP35 in a model cellular setting, selectively covalently modify C106, and exhibit antiplasmodium activity in blood-stage cultured parasites.

PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5.

PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5. Current efforts are focused on understanding whether PRMT5-substrate adaptor disruption synergizes with immune therapies or new classes of PRMT5 inhibitors in CDKN2A/MTAP null cancer. Citation Format: Kathleen M. Mulvaney, Brian McMillan, Christa Blomquist, Nischal Acharya, Matthew Ranaghan, Devishi Kesar, Alessandra Ianari, William Sellers. PRMT5 substrate recruitment as a therapeutic target in CDKN2A/MTAP null cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 94.

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action and structure determination studies revealed that these compounds form a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts the PRMT5-RIOK1 complex, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive small molecule that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

FK506-binding protein 35, FKBP35, has been implicated as an essential malarial enzyme. Rapamycin and FK506 exhibit antiplasmodium activity in cultured parasites. However, due to the highly conserved nature of the binding pockets of FKBPs and the immunosuppressive properties of these drugs, there is a need for compounds that selectively inhibit FKBP35 and lack the undesired side effects. In contrast to human FKBPs, FKBP35 contains a cysteine, C106, adjacent to the rapamycin binding pocket, providing an opportunity to develop targeted covalent inhibitors of Plasmodium FKBP35. Here, we synthesize inhibitors of FKBP35, show that they directly bind FKBP35 in a model cellular setting, selectively covalently modify C106, and exhibit antiplasmodium activity in blood-stage cultured parasites.

Cerebellar neural stem cells (NSCs) require Hedgehog-Gli (Hh-Gli) signalling for their maintenance and Nanog expression for their self-renewal. To identify novel molecular features of this regulatory pathway, we used next-generation sequencing technology to profile mRNA and microRNA expression in cerebellar NSCs, before and after induced differentiation (Diff-NSCs). Genes with higher transcript levels in NSCs (vs. Diff-NSCs) included Foxm1, which proved to be directly regulated by Gli and Nanog. Foxm1 in turn regulated several microRNAs that were overexpressed in NSCs: miR-130b, miR-301a, and members of the miR-15~16 and miR-17~92 clusters and whose knockdown significantly impaired the neurosphere formation ability. Our results reveal a novel Hh-Gli-Nanog-driven Foxm1-microRNA network that controls the self-renewal capacity of NSCs.

We describe a large outbreak associated with a crusted (Norwegian) scabies case in an immunocompromised patient following treatment with TNF-α inhibitor (adalimumab) for psoriasis arthritis. The increasing use of TNF-α inhibitors should induce clinicians to consider this serious parasitic infection when evaluating skin rashes in patients receiving biologic therapies.Infect. Control Hosp. Epidemiol. 2015;36(11):1358–1360

Summary The retinoblastoma protein (pRB) tumor suppressor blocks cell proliferation by repressing the E2F transcription factors. This inhibition is relieved through mitogen-induced phosphorylation of pRB, triggering E2F release and activation of cell-cycle genes. E2F1 can also activate proapoptotic genes in response to genotoxic or oncogenic stress. However, pRB's role in this context has not been established. Here we show that DNA damage and E1A-induced oncogenic stress promote formation of a pRB-E2F1 complex even in proliferating cells. Moreover, pRB is bound to proapoptotic promoters that are transcriptionally active, and pRB is required for maximal apoptotic response in vitro and in vivo. Together, these data reveal a direct role for pRB in the induction of apoptosis in response to genotoxic or oncogenic stress.

E2F1, a member of the E2F family of transcription factors, in addition to its established proliferative effect, has also been implicated in the induction of apoptosis through p53-dependent and p53-independent pathways. Several genes involved in the activation or execution of the apoptotic programme have recently been shown to be upregulated at the transcriptional level by E2F1 overexpression, including the genes encoding INK4a/ARF, Apaf-1, caspase 7 and p73 (refs 3-5). E2F1 is stabilized in response to DNA damage but it has not been established how this translates into the activation of specific subsets of E2F target genes. Here, we applied a chromatin immunoprecipitation approach to show that, in response to DNA damage, E2F1 is directed from cell cycle progression to apoptotic E2F target genes. We identify p73 as an important E2F1 apoptotic target gene in DNA damage response and we show that acetylation is required for E2F1 recruitment on the P1p73 promoter and for its transcriptional activation.

The retinoblastoma protein gene RB-1 is mutated in one-third of human tumors. Its protein product, pRB (retinoblastoma protein), functions as a transcriptional coregulator in many fundamental cellular processes. Here, we report a nonnuclear role for pRB in apoptosis induction via pRB's direct participation in mitochondrial apoptosis. We uncovered this activity by finding that pRB potentiated TNFα-induced apoptosis even when translation was blocked. This proapoptotic function was highly BAX-dependent, suggesting a role in mitochondrial apoptosis, and accordingly, a fraction of endogenous pRB constitutively associated with mitochondria. Remarkably, we found that recombinant pRB was sufficient to trigger the BAX-dependent permeabilization of mitochondria or liposomes in vitro. Moreover, pRB interacted with BAX in vivo and could directly bind and conformationally activate BAX in vitro. Finally, by targeting pRB specifically to mitochondria, we generated a mutant that lacked pRB's classic nuclear roles. This mito-tagged pRB retained the ability to promote apoptosis in response to TNFα and also additional apoptotic stimuli. Most importantly, induced expression of mito-tagged pRB in Rb−/−;p53−/− tumors was sufficient to block further tumor development. Together, these data establish a nontranscriptional role for pRB in direct activation of BAX and mitochondrial apoptosis in response to diverse stimuli, which is profoundly tumor-suppressive.

Checkpoint genes maintain genomic stability by arresting cells after DNA damage. Many of these genes also control cell cycle events in unperturbed cells. By conducting a screen for checkpoint genes in zebrafish, we found that dtl/cdt2 is an essential component of the early, radiation-induced G2/M checkpoint. We subsequently found that dtl/cdt2 is required for normal cell cycle control, primarily to prevent rereplication. Both the checkpoint and replication roles are conserved in human DTL. Our data indicate that the rereplication reflects a requirement for DTL in regulating CDT1, a protein required for prereplication complex formation. CDT1 is degraded in S phase to prevent rereplication, and following DNA damage to prevent origin firing. We show that DTL associates with the CUL4–DDB1 E3 ubiquitin ligase and is required for CDT1 down-regulation in unperturbed cells and following DNA damage. The cell cycle defects of Dtl-deficient zebrafish are suppressed by reducing Cdt1 levels. In contrast, the early G2/M checkpoint defect appears to be Cdt1-independent. Thus, DTL promotes genomic stability through two distinct mechanisms. First, it is an essential component of the CUL4–DDB1 complex that controls CDT1 levels, thereby preventing rereplication. Second, it is required for the early G2/M checkpoint.

E2F1, a member of the E2F family of transcription factors, plays a pivotal role in controlling both physiological cell-cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. In response to genotoxic stresses, E2F1 is stabilized by signals that include ATM-dependent phosphorylation. We recently demonstrated that DNA damage induces also E2F1 acetylation, which is required for its recruitment onto apoptotic gene promoters. Here we show that E2F1 is stabilized in response to doxorubicin and cisplatin treatments even in the absence of either ATM-dependent phosphorylation or p53 and cAbl, two major transducers of DNA damage signaling. We found that acetylation of E2F1 is, instead, required to stabilize the protein in response to doxorubicin. Finally, we report that the formation of E2F1-p300/CREB-binding protein-associated factor (P/CAF) complexes is preferentially induced in doxorubicin-treated cells, and that P/CAF acetyltransferase (HAT), but not p300 HAT activity, is required for a significant E2F1 stabilization and accumulation. Our results unveil a differential role of P/CAF and p300 in acetylation-induced stabilization of E2F1, thus supporting a specific role for P/CAF HAT activity in E2F1-dependent apoptosis in response to DNA damage.

To determine the concordance rate for Type II (non-insulin-dependent) diabetes mellitus in monozygotic twin pairs, initially ascertained discordant for diabetes, we carried out a prospective study on 44 non-diabetic subjects, each of whom had a sibling twin with diabetes (21 men, 23 women, median age 55 years, interquartile range 47–65). The subjects were referred as discordant for Type II diabetes. The twin pairs were part of the British Diabetic Twin Study and ascertained between May 1968 and January 1998. These subjects underwent an OGTT at time of referral and periodically thereafter. The mean follow-up was 8 years (range 0–18 years) and data were collected until January 1996. The percentage of twins who developed Type II diabetes was assessed by standard actuarial life-table methods and the pairwise concordance rate, that is the proportion of concordant pairs over the sum of concordant and discordant pairs, was calculated. The observed rates of concordance for Type II diabetes at 1, 5, 10, and 15 years follow-up were 17, 33, 57, and 76 %, respectively. The concordance rate for any abnormality of glucose metabolism (either Type II diabetes or impaired glucose tolerance) at 15 years follow-up was 96 %. The concordance rate for Type II diabetes in monozygotic twins is very high even in twins initially ascertained discordant for diabetes. [Diabetologia (1999) 42: 146–150]

Retroperitoneal soft-tissue sarcomas are a heterogeneous group of rare and peculiar mesenchymal tumors. They are locally invasive and have a peak incidence in the fifth decade of life. They account for 0.1-0.2% of all solid tumors and 15% of all soft-tissue tumors. Liposarcomas are usually large and occur most frequently in the lower extremities, in the retroperitoneal, perineal and mesenteric region. In the retroperitoneum they grow slowly due to the ability of the abdominal cavity to accommodate these slowly expanding masses. They don't produce symptoms until they are very large and have invaded local tissues. The case of a 61-year old man with a retroperitoneal liposarcoma is reported. The tumor was discovered due to the association of abdominal mass, weight loss and persistent fever. The fever, especially, is present due to a wide tumor necrosis. The diagnosis was suggested by computed tomography. Normally, the interval between start of symptoms and diagnosis is included within three weeks and one year. Surgical complete resection of the mass with splenectomy and local postoperative radiotherapy were performed. The weight of the mass was 8.56 kilograms and the pathological evaluation showed a pleomorphic highly undifferentiated liposarcoma. This histological type normally presents many tumor giant cells, some of which have the features of lipoblasts. The single most important prognostic factor in patients with soft-tissue sarcomas is the histologic grade of the primary lesion. In the last AJCC Staging System the grades are assigned from grade 1 (well differentiated) to grade 3 (poorly differentiated). The present case is grade 3. In the treatment of sarcoma of the retroperitoneum or genitourinary tract, the conventional chemotherapy does not seem effective, while radiotherapy has a little improvement on survival. Local recurrences are frequent, especially in the first three years, often in the absence of distant metastases. When the tumor recurs locally, the best therapy is still to remove the mass. Sometimes, two or more operations may be necessary for the patient. Generally, the prognosis is poor with overall 5-year survival of 15-50%. The patient was admitted in our division 4 months after the first operation with poor medical condition. The patient died nine months after surgery.

Objectives To evaluate the sensitivity and specificity of the Bard BTA test compared with bladder washing cytology in patients with a history of transitional cell bladder cancer undergoing routine follow-up cystoscopy. Methods During routine follow-up for transitional cell bladder cancer, 75 patients underwent cystoscopy, bladder washing cytology, and the Bard BTA test, a latex agglutination test that qualitatively detects basement membrane complexes in voided urine. From October 1994 to October 1995, a total of 104 Bard BTA test examinations were performed. The results of the Bard BTA test were compared with those attained with cystoscopy and bladder washing cytology. Results Cystoscopy found tumors in 13 cases. The Bard BTA test was diagnostic in 7 (54%) cases; it was more sensitive than bladder washing cytology, which was positive in only 3 (23%) cases. However, the specificity of the Bard BTA was lower (9% clinically unconfirmed positive tests) than that attained with cytology. In 2 patients (2%) in whom the cystoscopy was negative, the Bard BTA test was predictive for a positive cystoscopy 3 and 5 months later. Conclusions The Bard BTA test is a noninvasive test that may be an important addition to cystoscopy and cytology in the routine surveillance of patients with a history of transitional cell cancer of the bladder.