HES6 is a novel member of the family of basic helix–loop–helix mammalian homologues of Drosophila Hairy and Enhancer of split. We have analyzed the biochemical and functional roles of HES6 in myoblasts. HES6 interacted with the corepressor transducin-like Enhancer of split 1 in yeast and mammalian cells through its WRPW COOH-terminal motif. HES6 repressed transcription from an N box–containing template and also when tethered to DNA through the GAL4 DNA binding domain. On N box–containing promoters, HES6 cooperated with HES1 to achieve maximal repression. An HES6–VP16 activation domain fusion protein activated the N box–containing reporter, confirming that HES6 bound the N box in muscle cells. The expression of HES6 was induced when myoblasts fused to become differentiated myotubes. Constitutive expression of HES6 in myoblasts inhibited expression of MyoR, a repressor of myogenesis, and induced differentiation, as evidenced by fusion into myotubes and expression of the muscle marker myosin heavy chain. Reciprocally, blocking endogenous HES6 function by using a WRPW-deleted dominant negative HES6 mutant led to increased expression of MyoR and completely blocked the muscle development program. Our results show that HES6 is an important regulator of myogenesis and suggest that MyoR is a target for HES6-dependent transcriptional repression.
Recent studies have disclosed higher rates of infection in patients with the classic Philadelphia-negative myeloproliferative neoplasms (MPN).1-3 Importantly, this increased propensity to infections appears to be inherent to the disease itself, irrespective of cytoreductive therapy.2, 3 The risk of death due to infection is also increased in the MPN population compared to controls.2, 4 From a real-world vantage point, an informative large-scale European study recently found relatively high rates of infection (50.5%) in their MPN cohort, more pronounced in those patients with myelofibrosis (MF) and/or receiving ruxolitinib (RUX) therapy.1 However, further contemporary data examining the direct impact of MPN subtype, driver mutation, disease-directed therapy, prophylaxis and vaccination, and select concurrent medication on infection rates in MPN remain scarce. No such studies have been conducted, to our knowledge, in Canada, and few – if any, in North America. Moreover, the rates of Covid-19 infection and associated clinical consequences in a large unselected, real-world MPN cohort have not previously been assessed. The objectives of this pan-provincial population-based study were accordingly to evaluate: (i) infection frequency and types; (ii) common prophylactic measures; and (iii) influence of molecular background, disease-directed therapy, and concomitant medications, on infection outcomes in patients with Philadelphia-negative MPN included in an extensive Quebec registry. The current prospective study included five academic centers across Quebec, Canada. The diagnosis of polycythemia vera (PV), essential thrombocythemia (ET) and MF were in concordance with the 2016 WHO criteria.5 All patients, having given prior informed consent, were participants in the chronic myeloid leukemia-MPN Quebec Research Group (GQR LMC-NMP) registry and database. Data was collected between June 29, 2020 and August 3, 2020 and consisted of patient-reported details, via structured telephone and/or email questionnaires, with subsequent physician review of the nature/frequency of infection, vaccination and drug prophylaxis, and MPN-directed drug exposure in the past 12 months. The MPN-specific data (for example, subtype, driver mutations status,) were abstracted from database records. Infection-associated data related exclusively to the previous 12-month time period. Standard statistical methods were used to compare variables across groups with p values < .05 considered significant. Differences in the distribution of continuous variables between categories were compared using the Mann–Whitney or Kruskal–Wallis test. Categorical variables were compared using the χ2 test. The JMP Pro 14.1.0 software package was used for all analyses (SAS Institute, Cary, NC, USA). The clinical characteristics and infection types and severity for 257 informative MPN cases stratified by disease subtype are presented in Table 1. The cohort included 95 PV, 125 ET, and 37 MF patients with overall median age of 70 years (range 26–93 years) and a 40:60 male to female distribution. Driver mutation status for 235 evaluable cases was as follows: JAK2 (84%), CALR (12%), MPL (2%), and triple negative (2%). In the previous 12 months, the majority of patients had been exposed to hydroxyurea (HU) (n = 155; 68%), followed by RUX (n = 32; 14%), multiple cytoreductive drugs or combinations (n = 18; 8%), anagrelide (n = 8; 3%), interferon (n = 4; 2%), or busulfan (n = 2; 1%), while 10 patients (4%) had not received any MPN-specific therapy. Most subjects had received low-dose aspirin (n = 201; 78%), with skewing towards PV/ET versus MF cohorts (p < .001). Overall, 86 patients (33%) reported at least one infectious episode over the past 12 months. For those treated on an outpatient basis, 48 (19%) reported a single episode while 29 (11%) had more than one episode, with relatively balanced distribution across MPN subtypes (p = .6). In total, 12 patients (5%) required inpatient treatment, represented primarily by MF patients (n = 5; 14%) versus those with PV (n = 3; 3%) and ET (n = 4; 3%) (p = .06). Infection types overall were as follows: urinary tract (11%), ears nose throat (ENT) (9%), other – including dental, vaginal, and epididymal (7%), cutaneous (6%), herpes zoster (5%), bronchitis (4%), pneumonia (3%), gastrointestinal (GI) (2%), and Covid-19 (1%). Infection types were proportional across MPN subtypes with the exception of a trend towards higher rates of zoster infection in the MF sub-group (14% vs. 3% PV and 4% ET; p = .08). Antibiotic, antiviral, and antifungal prophylaxis had been received by 3 (1%), 3 (1%), and 1 (<1%) patient, respectively, with the majority consisting of MF patients. Vaccinations for influenza, herpes zoster, and pneumococcal pneumonia had been reported in 115 (45%), 16 (6%), and 19 (7%) patients, respectively, with no bias in MPN type. Clinical parameters of MPN patients stratified according to occurrence of infectious complications are detailed in Table S1. No appreciable differences were detected on the basis of age (>65 years old vs. younger; p = .4), MPN subtype (p = .6), driver mutation (p = .9), or concurrent aspirin therapy (p = .3). Analysis by gender disclosed a higher frequency of infections in females versus males (38% vs. 26%; p = .04). From a treatment standpoint, most of the patients treated with ruxolitinib in the past 12 months presented at least one infectious event (n = 18; 56%), while events were reported in 39%, 30%, and 13% of those having received multiple/combination therapies, hydroxyurea, and anagrelide, respectively. As 18 patients (8%) had received either multiple agents or combinations thereof, specification of any RUX use in the past 12 months disclosed a significant association with infectious events (p = .001), while none were observed with hydroxyurea use (p = .2). Of note, none of the four patients treated with interferon and, conversely, both patients treated with busulfan, reported an infectious episode. While only a minority of patients were exempt from any cytoreductive therapy in the previous year (n = 10), 20% of them reported being treated for an infection. Vaccination status for influenza and herpes zoster showed no significant association with infection rate (p = .2 and .4, respectively), though none of the 16 patients vaccinated against zoster reported zoster infections in the timeframe of interest. In contrast, the majority of patients having received the pneumococcal vaccine (n = 11; 58%) declared having had an infectious event in the past year (p = .02). Of the entire cohort, only two patients reported having contracted the Covid-19 virus, one of which required hospitalization without intensive care support. This multicenter prospective, patient-reported study of infection outcomes in MPN confirms clinically relevant rates of infection in all MPN subtypes, though somewhat lower than those previously described (33% vs. previously reported 45–50%).1, 3 Though limited by factors including potential recall and regional biases, as well as logistics beyond the scope of this report, such as disease and therapy duration, co-morbidities, complete drug history, or infection grade, the current study critically represents a large dataset derived from an "all-comers" highly inclusive MPN registry, circumventing the bias towards reporting solely severe infections/hospitalizations, and addresses fundamental questions about the prevalent phenomenon of infections in MPN from a practical, real-world standpoint. Interestingly, our data did not disclose a significant association between infection risk and advanced age, a finding which concurs with some,1 though not other reports,2 suggesting an uncertain role for age as a substantive, isolated risk factor for infections in MPN. The apparent increase in infection rate in women versus men in this study is challenging to interpret, but may be due to gender differences in adverse event reporting, known to occur in some patient-reported study settings, and possibly disproportionate representation of UTI in the range of infections. Importantly, in contrast with another similarly-designed study,1 while more MF patients experienced infectious events compared to their PV/ET counterparts, this did not meet statistical significance, suggesting that in our cohorts, other variables (e.g., disease duration, risk category, or perhaps newly-instituted public health pandemic restrictions and practices) may have more markedly influenced outcomes. Consistent with previous reports, exposure to RUX significantly increased the risk of infection in MPN patients, with a specific predilection towards herpes infections,6 while exposure to hydroxyurea had no impact, conceivably due, in part, to its use primarily in PV/ET populations. The former observation should sensitize physicians and patients to the infectious risks associated with RUX and advocates for potentially accrued infection surveillance of MPN patients treated with this agent. Interestingly, while uncommonly utilized, individuals treated with busulfan experienced the second highest rate of infection, while those having been exposed to multiple or combination therapies were the third most affected, serving as a potential warning when considering multi-drug regimens in these patients. Finally, patients who were vaccinated for pneumococcus in the last 12 months had a significant increase in infectious events, possibly reflecting an impetus for vaccination driven by recent infections or perhaps greater inherent susceptibility of these patients rationalizing the vaccination event. Of note, the fact that data collection occurred during, and pertained to (in part), the period affected by the global Covid-19 pandemic, greater caution may have been exercised by these patients, potentially affecting infection endpoints. As certain of our observations challenge data from previous reports1 (e.g., lower rates of infection, lack of association with MPN sub-type), highlighted by the fact that few patients in our cohort contracted Covid-19 (much less than would be expected given provincial infection rates and the median age of our cohort), the far-reaching impact of the pandemic and associated sanitary measures may at least be partially accountable, which in itself is a novel finding mandating further study. If rates of infections in patients with MPN have in fact been reduced since the onset of the Covid-19 outbreak, this data provide a clear signal to both patients and health care providers that current precautions are effective and warranted. Overall, the current observations provide additional significant insight into infection outcomes in MPN and represent the only such data from a large provincial registry reflecting a real-world setting. None. Data available on request Table S1. MPN patient characteristics stratified by the occurrence of infectious complications Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
In the version of this Article initially published, a versioning error led to a mistake in the third paragraph of the Discussion.In the text now reading "In our dataset, the most extreme and sustained increase in SARS-CoV-2 cases associated with school opening was in the South, where school opening was associated with a weekly increase in cases ranging from 9.8 to 21.3 per 100,000 people, " the range initially reported was "7.8 to 18.9 per 100,000." The results presented in the text and figures are unaffected.
A81 GMX1777 (EB1627) is a soluble prodrug of the pharmacologically active compound GMX1778 (CHS828), and is currently in Phase I clinical trials. The mechanism of action of this small molecule with potent and broad antitumor activity was believed to involve NF-κB inhibition. We now report that GMX1778 functions by inhibiting nicotinamide phosphoribosyl transferase (NAMPRT), an enzyme involved in nicotinamide adenine dinucleotide (oxidized) (NAD+) biosynthesis and that NF-κB inhibition is only a consequence of NAD+ decline. To determine the primary mechanism of action of GMX1778, a combination of global metabolomics and targeted biochemical pathway profiling studies were undertaken to track intracellular physiological changes over time. Of the limited physiological changes in IM-9 cells observed after treatment with GMX1778 for 6 hours, there occurred a 60% decline of intracellular NAD+ levels, with a 91% decline noted at 13 hours. NAD+ decline is followed by a gradual decline of ATP and cell death 48 to 72 hours post treatment. Addition of nicotinamide or nicotinic acid completely rescued GMX1778-treated HeLa cells from NAD+ decline, NF-κB inhibition and cell death; thereby substantiating that lack of NAD+ is the cause of GMX1778-induced NF-κB inhibition and cytotoxicity. Modulating NAD+ levels in a similar manner did not affect the activity of an unrelated cytotoxic agent 5-FU. Metabolic experiments using labeled precursors showed that GMX1778 inhibits the generation of NAD+ from 14C nicotinamide but not from 14C nicotinic acid in HeLa cells and cell extracts. Further experiments using purified human enzyme demonstrated that GMX1778 inhibits NAMPRT, the rate-limiting enzyme that converts nicotinamide (NAM) to nicotinamide mononucleotide (NAMN); the apparent Ki (1-3 nM) of GMX1778 for NAMPRT correlates well with the low nanomolar IC50 of this compound in multiple human cell lines. In vitro cytotoxicity studies showed a positive correlation between the level of NAMPRT expression and sensitivity to GMX1778. In support of these results, partial siRNA knockdown of NAMPRT in HeLa cells sensitized cells to GMX1778. Together these results demonstrate that GMX1778 is a potent and specific inhibitor of NAMPRT, resulting in a rapid drop in cellular NAD+. Tumor cells have elevated NAMPRT and a high rate of NAD+ turnover due to high ADP-ribosylation activity required for DNA repair, genome stability, telomere maintenance making them more susceptible to NAMPRT inhibition than normal cells. This novel mechanism supports the clinical use of GMX1777 as an anti-cancer agent and provides a rationale for the use of GMX1777 in combination with DNA damaging agents for future trials.
Failure to achieve early molecular response (EMR, BCR-ABL1 copies ≤ 10% by the international scale) 3 months after initiation of tyrosine kinase inhibitor (TKI) therapy has been shown to be a poor prognostic marker in chronic myeloid leukemia in chronic phase (CML-CP).1 However, whether the risk of adverse outcomes of EMR failure can be reduced by a switch in TKI therapy and when the switch should occur are unknown. Furthermore, patients with EMR failure may be a heterogeneous group that can be further stratified by long and short BCR-ABL1 halving time (HT).2 Thus, patients whose BCR-ABL1 copy numbers at 3 months are halved or near-EMR may not be need an early switch.3 We conducted an observational study to address the role of early switch of TKI therapy in EMR failure and long HT. We evaluated the association of switch within 6 months to second-line TKI for EMR failure with the achievement of major molecular response (MMR, BCR-ABL1 copies ≤ 0.1% by the international scale) among patients enrolled in the Quebec CML Registry (see detailed methods in the Supporting Information). Among 283 evaluable patients, 59 (20.8%) failed to achieve EMR while this milestone was achieved in 224. Baseline characteristics of patients with and without EMR were comparable (Table S1 in the Supporting Information). EMR failure was associated with a reduced probability of MMR (adjusted hazard ratio [HR], 0.46; 95% confidence intervals [CI], 0.33–0.63). In the subset with EMR failure, early switch occurred in 23 (39.0%) patients, while 36 (61.0%) remained on frontline TKI through 6 months from EMR failure. Compared with non-early switchers, early switchers were more likely to be male, to have received imatinib in the front line and to have been treated at lower volume centers (Table S2 in the Supporting Information). Patients with EMR failure were followed for a mean (standard deviation) of 19.1 (15.0) months from EMR determination. During 1125 months of follow-up, 45 patients achieved MMR. To avoid immortal time bias, we treated the switch to second-line TKI as a time-updated covariate such that patients were considered as “no switch” up to the time a switch occurred. The HR for achieving MMR was adjusted for age, sex, and first-line TKI (imatinib vs non-imatinib). The adjusted HR for MMR with early switch at 6 months was 1.63 (95% CI, 0.83–3.23), compared with no early switch. The adjusted HR for MMR with a switch within 3 months was similar (HR 1.47; 95% CI, 0.70–3.09), but was below the null with a switch up to 12 months (HR 0.79; 95% CI, 0.41–1.54) (Table 1). Among the 59 patients with EMR failure, baseline BCR-ABL1 copy number was available for 46. The median HT in this population was 65 days using the method suggested by Branford et al.2 The adjusted HR for MMR with early switch within 6 months in patients with short HT was 0.63 (95% CI, 0.15–2.60), while in patients with long HT it was 2.40 (95% CI, 0.66–8.71) (Table 1). Finally, after entering all the covariates in the model in a sensitivity analysis, the adjusted HR for MMR with early switch was 2.06 (95% CI, 0.85–4.98). Our study suggests that switch in TKI therapy within 6 months is associated with a higher likelihood of achieving MMR in patients with EMR failure or long HT. We could not find any other studies reporting the rate of early switch among patients with EMR failure in routine CML care nor the comparative effectiveness of this approach. In our study, 17/23 (74%) of early switchers achieved MMR during follow-up, which compares favorably with the 21/54 (39%) who switched from imatinib to nilotinib in the Therapeutic Intensification in De Novo Leukaemia (TIDEL)-II trial.4 One possible explanation for this difference may come from the benefit suggested in this analysis to a switch no later than 6 months from EMR failure, whereas in TIDEL II there was heterogeneity in the timing of switch. The proportion of patients with EMR failure reported herein is very similar to that reported in several trials (18%).2 Further, the prognostic importance of EMR failure was reiterated in our study despite a surprisingly high rate of MMR (72.7% at 30 months) in the subset with EMR failure. This rate is higher than that reported in imatinib-treated patients (41% at 4 years),2 or in nilotinib-treated patients (29% at 2 years)5 enrolled in clinical trials. The strengths of our study include the use of time-dependent exposure definition, which avoids the misclassification of observation time in patients who switched at various time points after EMR failure. Further, we used only EMR determination using the international scale, which is comparable with trial populations and with the current standard of care. Lastly, we modeled early switch only in poor responders to frontline TKI and avoided comparisons with non-early switch in patients with good response, which would have led to confounding by indication, ie, comparing early switchers with patients who benefited from frontline TKI (which would have diluted the effect). Our study also has several limitations. In adjusted models, we were unable to demonstrate a statistically significant increase in MMR with early switch to second-line TKI, perhaps due to residual confounding in a heterogeneous population and small sample size. To avoid overfitting, we adjusted our primary analysis only for age, sex and first-line TKI. Notably, with inclusion of several additional covariates, the estimate for benefit with early switch only increased. Our sample size allowed us only to reject a probability lower than 0.83 (the lower CI limit), suggesting cautious interpretation of our findings. Lastly, we could not adjust our models for Sokal scores. Nevertheless, EMR failure and HT have been repeatedly shown to be more predictive of patient trajectories than Sokal.2, 6 In summary, this first study of the comparative effectiveness of early switch in EMR failure in routine care suggests that this strategy is associated with a greater likelihood of achieving MMR particularly in patients with long HT. Our findings provide a rationale to conduct a prospective investigation of early switch in this population. Study design: Kilil-Drori, Azoulay, Assouline Data analysis: Kilil-Drori, Yin Manuscript writing: All authors Data review: All authors Data collection: Harnois, Gratton, Del Corpo, Klil-Drori Obtained financial support: Assouline This work was supported in part by a grant from the Rossy Cancer Network to S.E. Assouline. A.J. Klil-Drori is supported by a fellowship grant from the Israel Cancer Research Fund. AJKD has received speaking honoraria from BMS. HJO has received speaking honoraria from Novartis, BMS, and Celgene. PL has received speaking honoraria from Novartis, Pfizer, BMS, and Paladin. LB has received speaking honoraria from Novartis, Pfizer, BMS, and Paladin. SEA has received speaking honoraria from Paladin, BMS and Pfizer. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD(+) biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD(+) turnover, which makes NAD(+) modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD(+) and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD(+) repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.
Abstract Background In the current study, the authors determined whether adhering to molecular monitoring guidelines in patients with chronic myeloid leukemia (CML) is associated with major molecular response (MMR) and assessed barriers to adherent monitoring. Methods Newly treated patients with CML from the Quebec province‐wide CML registry from 2005 to 2016 were included. Timely polymerase chain reaction (tPCR) was defined as the molecular assessment of BCR ‐ABL1 at the 3‐month, 12‐month, and 18‐month time points from the initiation of tyrosine kinase inhibitor (TKI) therapy. The cohort was analyzed as a nested case‐control study. Cases with a first‐ever MMR ( BCR‐ABL1 ≤0.1%, assessed at any time during follow‐up) were matched to up to 5 controls by duration of TKI therapy, volume of patients with CML at the treatment center, year of cohort entry, and age. Odds ratios (ORs) for the performance of tPCR and MMR were adjusted for sex, comorbidities, type of TKI, and other important covariates. Results The cohort included 496 patients. Of 392 MMR events, 67.9% occurred before 18 months. The performance of tPCR was associated with a doubling of the MMR rate (OR, 2.23; 95% confidence interval [95% CI], 1.56‐3.21) and was similar with 1 to 3 tPCRs performed ( P = .67). Furthermore, tPCRs at 3 months (OR, 2.77; 95% CI, 1.81‐4.23) and 12 months (OR, 3.00; 95% CI, 1.64‐5.49) were associated with achieving early MMR, whereas tPCRs at 18 months were not (OR, 1.23; 95% CI, 0.80‐1.89). Low‐volume centers were found to have lower adherence to tPCR (OR, 0.60; 95% CI, 0.40‐0.89). Conclusions Timely molecular assessment at 3 months and 12 months appears to benefit patients with CML. Adherence to timely monitoring should be encouraged, especially in low‐volume treatment centers.
