HDAC1,2 inhibition and doxorubicin impair Mre11-dependent DNA repair and DISC to override BCR-ABL1-driven DSB repair in Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia.

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms. The HDAC1,2 inhibitor but not doxorubicin alters nucleosomal occupancy to impact chromatin structure, as revealed by MNase-Seq. Quantitative mass spectrometry of the chromatin proteome along with functional assays showed that the HDAC1,2 inhibitor and doxorubicin either alone or in combination impair the central hub of DNA repair, the Mre11-Rad51-DNA ligase 1 axis, involved in BCR-ABL1-specific DSB repair signaling in Ph+ B-cell precursor ALL cells. HDAC1,2 inhibitor and doxorubicin interfere with DISC (DNA damage-induced transcriptional silencing in cis)) or transcriptional silencing program in cis around DSB sites via chromatin remodeler-dependent and -independent mechanisms, respectively, to further impair DSB repair. HDAC1,2 inhibitor either alone or when combined with doxorubicin decreases leukemia burden in vivo in refractory Ph+ B-cell precursor ALL patient-derived xenograft mouse models. Overall, our novel mechanistic and preclinical studies together demonstrate that HDAC1,2 selective inhibition can overcome DSB repair 'addiction' and provide an effective therapeutic option for Ph+ B-cell precursor ALL.

BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against mantle cell lymphoma cells.

Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins and nuclear factor-κB (NF-κB) target genes that undermines the growth and survival of mantle cell lymphoma (MCL) cells. However, BET bromodomain inhibitor (BETi) treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4 that potentially compromises the activity of BETi in MCL cells. Unlike BETi, BET-PROTACs (proteolysis-targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) recruit and utilize an E3-ubiquitin ligase to effectively degrade BETPs in MCL cells. BET-PROTACs induce more apoptosis than BETi of MCL cells, including those resistant to ibrutinib. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi, with depletion of c-Myc, CDK4, cyclin D1 and the NF-κB transcriptional targets Bcl-xL, XIAP and BTK, while inducing the levels of HEXIM1, NOXA and CDKN1A/p21. Treatment with ARV-771, which possesses superior pharmacological properties compared with ARV-825, inhibited the in vivo growth and induced greater survival improvement than the BETi OTX015 of immune-depleted mice engrafted with MCL cells. Cotreatment of ARV-771 with ibrutinib or the BCL2 antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of MCL cells. These studies highlight promising and superior preclinical activity of BET-PROTAC than BETi, requiring further in vivo evaluation of BET-PROTAC as a therapy for ibrutinib-sensitive or -resistant MCL.

Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells.

This corrects the article DOI: 10.1038/leu.2014.119.

Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary (s) AML cells.

The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Although the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reverse Phase Protein Array and mass cytometry 'CyTOF' analyses demonstrated that ARV-825 caused greater perturbations in messenger RNA (mRNA) and protein expressions than OTX015 in sAML cells. Specifically, compared with OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, p-STAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared with OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against established and PD CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

Pre-clinical efficacy of combined therapy with novel ß-catenin antagonist BC2059 and histone deacetylase inhibitor against AML cells.

The canonical wingless-type MMTV integration site (WNT)-ß-catenin pathway is essential for self-renewal, growth and survival of acute myeloid leukemia (AML) stem/blast progenitor cells (BPCs). Deregulated WNT signaling inhibits degradation of ß-catenin, causing increased nuclear translocation and co-factor activity of ß-catenin with the transcriptional regulator T-cell factor (TCF) 4/lymphoid enhancer factor 1 in AML BPCs. Here, we determined the pre-clinical anti-AML activity of the anthraquinone oxime-analog BC2059 (BC), known to attenuate ß-catenin levels. BC treatment disrupted the binding of ß-catenin with the scaffold protein transducin ß-like 1 and proteasomal degradation and decline in the nuclear levels of ß-catenin. This was associated with reduced transcriptional activity of TCF4 and expression of its target genes, cyclin D1, c-MYC and survivin. BC treatment dose-dependently induced apoptosis of cultured and primary AML BPCs. Treatment with BC also significantly improved the median survival of immune-depleted mice engrafted with either cultured or primary AML BPCs, exhibiting nuclear expression of ß-catenin. Co-treatment with the pan-histone deacetylase inhibitor panobinostat and BC synergistically induced apoptosis of cultured and primary AML BPCs, including those expressing FLT3-ITD, as well as further significantly improved the survival of immune-depleted mice engrafted with primary AML BPCs. These findings underscore the promising pre-clinical activity and warrant further testing of BC against human AML, especially those expressing FLT3-ITD.

Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells.

The histone demethylase LSD1 (KDM1A) demethylates mono- and di-methylated (Me2) lysine (K) 4 on histone H3. High LSD1 expression blocks differentiation and confers a poor prognosis in acute myeloid leukemia (AML). Here, treatment with the novel LSD1 antagonist SP2509 attenuated the binding of LSD1 with the corepressor CoREST, increased the permissive H3K4Me3 mark on the target gene promoters, and increased the levels of p21, p27 and CCAAT/enhancer binding protein α in cultured AML cells. In addition, SP2509 treatment or LSD1 shRNA inhibited the colony growth of AML cells. SP2509 also induced morphological features of differentiation in the cultured and primary AML blasts. SP2509 induced more apoptosis of AML cells expressing mutant NPM1 than mixed-lineage leukemia fusion oncoproteins. Treatment with SP2509 alone significantly improved the survival of immune-depleted mice following tail-vein infusion and engraftment of cultured or primary human AML cells. Co-treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) and SP2509 was synergistically lethal against cultured and primary AML blasts. Compared with each agent alone, co-treatment with SP2509 and PS significantly improved the survival of the mice engrafted with the human AML cells, without exhibiting any toxicity. Collectively, these findings show that the combination of LSD1 antagonist and pan-HDI is a promising therapy warranting further testing against AML.

Phase Ia/II, two-arm, open-label, dose-escalation study of oral panobinostat administered via two dosing schedules in patients with advanced hematologic malignancies.

Panobinostat is a potent oral pandeacetylase inhibitor that leads to acetylation of intracellular proteins, inhibits cellular proliferation and induces apoptosis in leukemic cell lines. A phase Ia/II study was designed to determine the maximum-tolerated dose (MTD) of daily panobinostat, administered on two schedules: three times a week every week or every other week on a 28-day treatment cycle in patients with advanced hematologic malignancies. The criteria for hematologic dose-limiting toxicities differed between patients with indications associated with severe cytopenias at baseline (leukemia and myeloid disorders) and those less commonly associated with baseline cytopenias (lymphoma and myeloma). In patients with leukemia and myeloid disorders, 60 mg was the MTD for weekly as well as biweekly panobinostat. In patients with lymphoma and myeloma, 40 mg was the recommended dose for phase II evaluation (formal MTD not determined) of weekly panobinostat, and 60 mg was the MTD for biweekly panobinostat. Overall, panobinostat-related grade 3-4 adverse events included thrombocytopenia (41.5%), fatigue (21%) and neutropenia (21%). Single-agent activity was observed in several indications, including Hodgkin lymphoma and myelofibrosis. This phase Ia/II study provided a broad analysis of the safety profile and efficacy of single-agent panobinostat in patients with hematologic malignancies.

Prediction of outcomes in patients with Ph+ chronic myeloid leukemia in chronic phase treated with nilotinib after imatinib resistance/intolerance.

The purpose was to assess predictive factors for outcome in patients with chronic myeloid leukemia (CML) in chronic phase (CML-CP) treated with nilotinib after imatinib failure. Imatinib-resistant and -intolerant patients with CML-CP (n=321) were treated with nilotinib 400 mg twice daily. Of 19 baseline patient and disease characteristics and two response end points analyzed, 10 independent prognostic factors were associated with progression-free survival (PFS). In the multivariate analysis, major cytogenetic response (MCyR) within 12 months, baseline hemoglobin ≥ 120 g/l, baseline basophils <4%, and absence of baseline mutations with low sensitivity to nilotinib were associated with PFS. A prognostic score was created to stratify patients into five groups (best group: 0 of 3 unfavorable risk factors and MCyR by 12 months; worst group: 3 of 3 unfavorable risk factors and no MCyR by 12 months). Estimated 24-month PFS rates were 90%, 79%, 67% and 37% for patients with prognostic scores of 0, 1, 2 and 3, respectively, (no patients with score of 4). Even in the presence of poor disease characteristics, nilotinib provided significant clinical benefit in patients with imatinib-resistant or -intolerant CML. This system may yield insight on the prognosis of patients.

Phase I-II study of vorinostat plus paclitaxel and bevacizumab in metastatic breast cancer: evidence for vorinostat-induced tubulin acetylation and Hsp90 inhibition in vivo.

In preclinical models, the histone deacetylase inhibitor vorinostat sensitizes breast cancer cells to tubulin-polymerizing agents and to anti-vascular endothelial growth factor-directed therapies. We sought to determine the safety and efficacy of vorinostat plus paclitaxel and bevacizumab as first-line therapy in metastatic breast cancer (MBC), and the biological effects of vorinostat in vivo. For this purpose of this study, 54 patients with measurable disease and no prior chemotherapy for MBC received vorinostat (200 or 300 mg PO BID) on days 1-3, 8-10, and 15-17, plus paclitaxel (90 mg/m(2)) on days 2, 9, 16, and bevacizumab (10 mg/kg) on days 2 and 16 every 28 days. The primary objective of the phase I study was to determine the recommended phase II dose (RPTD) of vorinostat, and for the phase II to detect an improvement of response rate from 40 to 60% (alpha = 0.10, beta = 0.10). No dose limiting toxicities were observed, and the RPTD of vorinostat was 300 mg BID. For the primary efficacy analysis in 44 patients at the RPTD, we observed 24 objective responses (55%, 95% confidence intervals (C.I) 39%, 70%). The adverse event profile was consistent with paclitaxel-bevacizumab, with the exception of increased diarrhea with the addition of vorinostat. Analysis of serial tumor biopsies in seven patients showed increased acetylation of Hsp90 and α-tubulin following vorinostat. Vorinostat induces histone and alpha tubulin acetylation and functional inhibition of Hsp90 in breast cancer in vivo and can be safely combined with paclitaxel and bevacizumab.

Incidence of road injuries in Mexico: country report.

We used data from various sources to triangulate to a national snapshot of the incidence of fatal and non-fatal road traffic injuries in Mexico in 2005. Data sources used include national death registration data, national hospital discharge data and a nationally representative health survey. We estimate that in 2005, 19,389 people died due to injuries and nearly one million were injured in road traffic crashes. While deaths in high-income countries are declining, this is not the case in Mexico. Young adult males are the demographic at the highest risk in non-fatal crashes, but the elderly have the highest road death rates primarily due to pedestrian crashes. Pedestrians alone comprise nearly half (48%) of all deaths. Cars pose a substantial threat to occupants (38% of deaths and 39% of hospital admissions) and to other road users.

Phase I study of the heat shock protein 90 inhibitor alvespimycin (KOS-1022, 17-DMAG) administered intravenously twice weekly to patients with acute myeloid leukemia.

Heat shock protein 90 (Hsp90) is a molecular chaperone with many oncogenic client proteins. The small-molecule Hsp90 inhibitor alvespimycin, a geldanamycin derivative, is being developed for various malignancies. This phase 1 study examined the maximum-tolerated dose (MTD), safety and pharmacokinetic/pharmacodynamic profiles of alvespimycin in patients with advanced acute myeloid leukemia (AML). Patients with advanced AML received escalating doses of intravenous alvespimycin (8-32 mg/m(2)), twice weekly, for 2 of 3 weeks. Dose-limiting toxicities (DLTs) were assessed during cycle 1. A total of 24 enrolled patients were evaluable for toxicity. Alvespimycin was well tolerated; the MTD was 24 mg/m(2) twice weekly. Common toxicities included neutropenic fever, fatigue, nausea and diarrhea. Cardiac DLTs occurred at 32 mg/m(2) (elevated troponin and myocardial infarction). Pharmacokinetics revealed linear increases in C(max) and area under the curve (AUC) from 8 to 32 mg/m(2) and minor accumulation upon repeated doses. Pharmacodynamic analyses on day 15 revealed increased apoptosis and Hsp70 levels when compared with baseline within marrow blasts. Antileukemia activity occurred in 3 of 17 evaluable patients (complete remission with incomplete blood count recovery). The twice-weekly administered alvespimycin was well tolerated in patients with advanced AML, showing linear pharmacokinetics, target inhibition and signs of clinical activity. We determined a recommended phase 2 dose of 24 mg/m(2).

Building national estimates of the burden of road traffic injuries in developing countries from all available data sources: Iran.

OBJECTIVE: To use a range of existing information sources to develop a national snapshot of the burden of road traffic injuries in one developing country-Iran. METHODS: The distribution of deaths was estimated by using data from the national death registration system, hospital admissions and outpatient visits from a time-limited hospital registry in 12 of 30 provinces, and injuries that received no institutional care using the 2000 demographic and health survey. Results were extrapolated to national annual incidence of health burden differentiated by age, sex, external cause, nature of injuries and institutional care. RESULTS: In 2005, 30,721 Iranians died annually in road traffic crashes and over one million were injured. The death rate (44 per 100,000) is the highest of any country in the world for which reliable estimates are available. Road traffic injuries are the third leading cause of death in Iran. While young adults are at high risk in non-fatal crashes, the elderly have the highest total death rates, largely due to pedestrian crashes. While car occupants lead the death count, motorized two-wheeler riders dominate hospital admissions, outpatient visits and health burden. CONCLUSIONS: Reliable estimates of the burden of road traffic injuries are an essential input for rational priority setting. Most low income countries are unlikely to have national injury surveillance systems for several decades. Thus national estimates of the burden of injuries should be built by collating information from all existing information sources by appropriately correcting for source specific shortcomings.

Targeting HSP90 for cancer therapy.

Heat-shock proteins (HSPs) are molecular chaperones that regulate protein folding to ensure correct conformation and translocation and to avoid protein aggregation. Heat-shock proteins are increased in many solid tumours and haematological malignancies. Many oncogenic proteins responsible for the transformation of cells to cancerous forms are client proteins of HSP90. Targeting HSP90 with chemical inhibitors would degrade these oncogenic proteins, and thus serve as useful anticancer agents. This review provides an overview of the HSP chaperone machinery and the structure and function of HSP90. We also highlight the key oncogenic proteins that are regulated by HSP90 and describe how inhibition of HSP90 could alter the activity of multiple signalling proteins, receptors and transcriptional factors implicated in carcinogenesis.

Fifteen-year median follow-up results after neoadjuvant doxorubicin, followed by mastectomy, followed by adjuvant cyclophosphamide, methotrexate, and fluorouracil (CMF) followed by radiation for stage III breast cancer: a phase II trial (CALGB 8944).

PURPOSE: To describe long-term results of a multimodality strategy for stage III breast cancer utilizing neoadjuvant doxorubicin followed by mastectomy, CMF, and radiotherapy. PATIENTS AND METHODS: Women with biopsy-proven, clinical stage III breast cancer and adequate organ function were eligible. Neoadjuvant doxorubicin (30 mg/m(2) days 1-3, every 28 days for 4 cycles) was followed by mastectomy, in stable or responding patients. Sixteen weeks of postoperative CMF followed (continuous oral cyclophosphamide (2 mg/kg/day); methotrexate (0.7 mg/kg IV) and fluorouracil (12 mg/kg IV) weekly, weeks 1-8, and than biweekly, weeks 9-16). Radiation therapy followed adjuvant chemotherapy. RESULTS: Clinical response rate was 71% (79/111, 95% CI = 62-79%), with 19% complete clinical response. Pathologic complete response was 5% (95% CI = 2-11%). Median follow-up is 15.6 years. Half of the patients progressed by 2.2 years; half died by 5.4 years (range 6 months-15 years). The hazard of dying was greatest in the first 5 years after diagnosis and declined thereafter. Time to progression and overall survival were predicted by number of pathologically involved lymph nodes (TTP: HR [10 vs. 1 node] 2.40, 95% CI = 1.63-3.53, P < 0.0001; OS: HR 2.50, 95% CI = 1.74-3.58, P < 0.0001). CONCLUSIONS: After multimodality treatment for locally advanced breast cancer, long-term survival was correlated with the number of pathologically positive lymph nodes, but not to clinical response. The hazard of death was highest during the first 5 years after diagnosis and declined thereafter, indicating a possible intermediate endpoint for future trials of neoadjuvant treatment.

Suppression of high-p{T} neutral pion production in central Pb+Pb collisions at sqrt[S{NN}]=17.3 GeV relative to p+C and p+Pb collisions.

Neutral pion transverse momentum spectra were measured in p+C and p+Pb collisions at sqrt[S{NN}]=17.4 GeV at midrapidity (2.3 less than or approximately equal eta{lab} less than or approximately equal 3.0) over the range 0.7 less than or approximately equal p{T} less than or approximately equal 3.5 GeV/c. The spectra are compared to pi{0} spectra measured in Pb+Pb collisions at sqrt[S{NN}]=17.3 GeV in the same experiment. For a wide range of Pb+Pb centralities (N{part} less than or approximately equal 300), the yield of pi{0}'s with p{T} greater than or approximately equal 2 GeV/c is larger than or consistent with the p+C or p+Pb yields scaled with the number of nucleon-nucleon collisions (N{coll}), while for central Pb+Pb collisions with N{part}greater than or approximately equal 350, the pi{0} yield is suppressed.

Community-based study on seroprevalence of herpes simplex virus type 2 infection in New Delhi.

PURPOSE: To determine the seroprevalence of herpes simplex virus type 2 (HSV-2) in two urban communities in Delhi and to correlate the presence of HSV-2 seroprevalence with sociodemographic profile, risk factors and presence of other reproductive tract infections (RTIs). METHODS: Men and women aged between 15-49 years from an urban slum and an urban middle class colony were invited to participate in the study. They provided interview information; blood for HSV-2, HIV and syphilis serology; first void urine specimens for diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis infection; and genital specimens for diagnosis of bacterial vaginosis, vaginal candidiasis and trichomoniasis. RESULTS: The prevalence of HSV-2 seropositivity was found to be 7 and 8.6% in men and women, respectively. HSV-2 seropositivity was found to be significantly associated with urban middle class community and older age. No statistically significant correlation was found between HSV-2 seropositivity and other laboratory-confirmed RTIs. CONCLUSIONS: The findings of our study indicate a relatively low prevalence of HSV-2 seropositivity and other sexually transmitted infections in the two communities that were studied.

Epidemilogical analysis of Neisseria gonorrhoeae isolates by antimicrobial susceptibility testing, auxotyping and serotyping.

PURPOSE: This study was carried out to analyze the epidemiology of gonorrhea based on antimicrobial susceptibility testing, auxotyping and serotyping in New Delhi, India. METHODS: Sixty gonococcal isolates from males with urethritis, females with endocervicitis and their sexual contacts were studied. The isolates were subjected to antimicrobial susceptibility testing, auxotyping and serotyping for epidemiological characterization. RESULTS: We observed nine antibiotic resistance patterns. Ninety-eight percent of isolates were resistant to ciprofloxacin, while 20% isolates were penicillinase producing N. gonorrhoeae (PPNG) and 18.3% isolates were tetracycline resistant N. gonorrhoeae (TRNG). Eight auxotypes were observed, of which the NR (non-requiring), proline requiring and arginine requiring were most common auxotypes. On the basis of serotyping alone, the gonococcal isolates could be differentiated into three serogroups and 18 serovars. Serogroup WI represented 46.7% and WII/III represented 51.7% of isolates and one strain was WI and WII/WIII serogroup combination. When results of auxotyping and serotyping were combined (A/S) 29 A/S classes could be identified. The most prevalent A/S classes were NR/Aost, NR/Arost, Pro/Aost and Pro/Boprt. CONCLUSIONS: Although A/S typing had the highest discriminatory index, isolates recovered from index case and their sexual contacts were found to be identical by all typing methods.