Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers.

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, McGuigan prodrugs were not. Other strategies, including cycloSal and lipid prodrugs of 5, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over 6. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation.

Circulating osteogenic progenitors and osteoclast precursors are associated with long-term glycemic control, sex steroids, and visceral adipose tissue in men with type 2 diabetes mellitus.

Introduction: Type 2 diabetes mellitus (T2DM) is well-known to be associated with normal bone density but, concurrently, low bone turnover and increased risk for fracture. One of the proposed mechanisms is possible derangement in bone precursor cells, which could be represented by deficiencies in circulating osteogenic progenitor (COP) cells and osteoclast precursors (OCP). The objective of our study is to understand whether extent of glycemic control has an impact on these cells, and to identify other factors that may as well. Methods: This was a secondary analysis of baseline data from 51 male participants, aged 37-65 in an ongoing clinical trial at Michael E. DeBakey VA Medical Center, Houston, Texas, USA. At study entry serum Hemoglobin A1c was measured by high-performance liquid chromatography osteocalcin (OCN) and C-terminal telopeptide of type 1 collagen (CTx) were measured by ELISA, and testosterone and estradiol by liquid-chromatography/mass-spectrometry. Areal bone mineral density (BMD), trabecular bone score and body composition were measured by dual energy x-ray absorptiometry, while COP and OCP were measured by flow cytometry. Results: When adjusted for serum testosterone, parathyroid hormone, and 25-hydroxyvitamin D, those with poor long-term glycemic control had significantly higher percentage of COP (p = 0.04). COP correlated positively with visceral adipose tissue (VAT) volume (r = 0.37, p = 0.01) and negatively with free testosterone (r = -0.28, p = 0.05) and OCN (r = -0.28, p = 0.07), although only borderline for the latter. OCP correlated positively with age, FSH, lumbar spine BMD, and COP levels, and negatively with glucose, triglycerides, and free estradiol. Multivariable regression analyses revealed that, in addition to being predictors for each other, another independent predictor for COP was VAT volume while age, glucose, and vitamin D for OCP. Conclusion: Our results suggest that high COP could be a marker of poor metabolic control. However, given the complex nature and the multitude of factors influencing osteoblastogenesis/adipogenesis, it is possible that the increase in COP is a physiologic response of the bone marrow to increased osteoblast apoptosis from poor glycemic control. Alternatively, it is also likely that a metabolically unhealthy profile may retard the development of osteogenic precursors to fully mature osteoblastic cells.

Baseline Testosterone Predicts Body Composition and Metabolic Response to Testosterone Therapy.

Context: Male hypogonadism adversely affects body composition, bone mineral density (BMD), and metabolic health. A previous report showed that pre-treatment testosterone (T) levels of <200 ng/dl is associated with greater improvement in spine BMD with T therapy. However, to date, there is no study that investigates whether baseline T levels also influence body composition and metabolic response to T therapy. Objective: The aim of this study is to determine if there are differences in the changes in body composition, metabolic profile, and bone turnover markers, in addition to BMD, in response to T therapy in men with a baseline T level of <264 ng/dl compared to those with levels ≥264 ng/dl. Methods: This is a secondary analysis of a single-arm, open-label clinical trial (NCT01378299) on pharmacogenetics of response to T therapy conducted between 2011 and 2016 involving 105 men (40-74 years old), with average morning T < 300 ng/dl, given intramuscular T cypionate 200 mg every 2 weeks for 18 months. Subjects were divided into those with baseline T levels of <264 ng/dl (N = 43) and those with ≥264 ng/dl (N = 57). T and estradiol (E2) were measured by liquid chromatography/mass spectrometry; serum bone turnover markers (C-telopeptide [CTX], osteocalcin, and sclerostin), adiponectin, and leptin were measured by enzyme-linked immunosorbent assay; glycated hemoglobin (HbA1c) was measured by high-performance liquid chromatography; and areal BMD and body composition was measured by dual-energy x-ray absorptiometry (DXA). Results: Men with T < 264 ng/dl showed greater increases in total fat-free mass (FFM) at 18 months compared to those with T ≥ 264 ng/dl (4.2 ± 4.1 vs. 2.7 ± 3.8%; p = 0.047) and unadjusted appendicular FFM at 6 and 18 months (8.7 ± 11.5 vs. 4.4 ± 4.3%, 7.3 ± 11.6 vs. 2.4 ± 6.8%; p = 0.033 and p = 0.043, respectively). Men with T ≥ 264 ng/dl showed significant decreases in HbA1c at 12 months (-3.1 ± 9.2 vs. 3.2 ± 13.9%; p = 0.005), fasting glucose at 18 months (-4.2 ± 31.9 vs. 13.0 ± 57.3%; p = 0.040), LDL at 6 months (-6.4 ± 27.5 vs. 12.8 ± 44.1%; p = 0.034), and leptin at 18 months (-40.2 ± 35.1 vs. -27.6 ± 31.0%; p = 0.034) compared to those with T < 264 ng/dl. No significant differences in BMD and bone turnover markers were observed. Conclusion: T therapy results in improvement in body composition irrespective of baseline T levels but T < 264 ng/dl is associated with greater improvement in FFM, whereas a T level of ≥264 ng/dl favors improvement in metabolic profile.

One-Year Mean A1c of > 7% is Associated with Poor Bone Microarchitecture and Strength in Men with Type 2 Diabetes Mellitus.

INTRODUCTION: Type 2 diabetes mellitus (T2DM) is associated with normal or slightly elevated bone mineral density (BMD) but paradoxically increased fracture risk. Although multiple mechanisms have been proposed to explain this observation, one thing is clear from prior studies, T2DM is associated with poor bone quality rather than a defect in bone quantity. The objective of our study is to evaluate the effect of longitudinal glycemic control on bone quality and bone turnover in men with T2DM. METHODS: This was a secondary analysis of baseline data from 169 male participants, aged 35-65 in 3 clinical trials. Participants were grouped according to the average of all their A1C measurements between 9 and 15 months prior to study entry (group 1: no T2DM, group 2: T2DM with A1C ≤ 7%, group 3: T2DM with A1C > 7%). At study entry serum osteocalcin and C-terminal telopeptide of type 1 collagen (CTx) were measured by ELISA, and testosterone and estradiol by liquid-chromatography/mass-spectrometry. Areal BMD, trabecular bone score and body composition were measured by dual-energy X-ray absorptiometry while volumetric BMD, bone microarchitecture, and bone strength were assessed by high-resolution peripheral quantitative computed tomography. RESULTS: At the tibia, trabecular separation was higher and trabecular number was significantly lower in group 3 compared to both groups 2 and 1, even after adjustments for covariates (p = 0.02 for both). Bone strength indices at the tibia such as stiffness and failure load were lowest in group 3, the difference being significant when compared to group 1 (p = 0.01, p = 0.009 respectively) but not to group 2, after adjustments for covariates. Bone turnover markers (osteocalcin and CTx) were significantly lower in group 3 relative to group 1, with CTx also being significantly lower in group 3 compared with group 2 (p < 0.001, p = 0.001 respectively). CONCLUSION: Poor glycemic control over the course of a year in men with T2DM is associated with poorer bone microarchitecture and strength, and reduced bone turnover. Conversely, good glycemic control in the setting of T2DM appears to attenuate this observed impairment in bone quality.

Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine.

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP's substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.

Hemoglobin A1c Threshold for Reduction in Bone Turnover in Men With Type 2 Diabetes Mellitus.

Background: Emerging data suggest that type 2 diabetes mellitus (T2D) is associated with an increased risk for fractures despite relatively normal or increased bone mineral density (BMD). Although the mechanism for bone fragility in T2D patients is multifactorial, whether glycemic control is important in generating this impairment in bone metabolism remains unclear. The purpose of our study is to identify a hemoglobin A1c (A1c) threshold level by which reduction in bone turnover begins in men with T2D. Method: A cross-sectional analysis of baseline data was obtained from 217 men, ages 35-65, regardless of the presence or absence of hypogonadism or T2D, who participated in 2 clinical trials. The following data were obtained: A1c by HPLC, testosterone and estradiol by LC/MS, bone turnover markers Osteocalcin [OC], C-terminal telopeptide [CTx], and sclerostin by ELISA, and BMD by DXA. Patients were grouped into 4 categories based of A1c (group I: <6%, group II: 6.0-6.4%, group III: 6.5-6.9%, and group IV: ≥7%). Threshold models were fit to the data using nonlinear regression and group comparisons among the different A1c categories performed by ANOVA. Results: Threshold model and nonlinear regression showed an A1c cut-off of 7.0, among all choices of A1cs, yields the least sum of squared errors. A comparison of bone turnover markers revealed relatively lower OC (p = 0.002) and CTx (p = 0.0002) in group IV (A1c ≥7%), compared to the other groups. An analysis of men with T2D (n = 94) showed relatively lower OC (p=0.001) and CTx (p=0.002) in those with A1c ≥7% compared to those with <7%, respectively. The significance between groups persisted even after adjusting for medications and duration of diabetes. Conclusion: An analysis across our entire study population showed a breakpoint A1c level of 7% or greater is associated with lower bone turnover. Also in men with T2D, an A1c ≥7% is associated with low bone turnover.

Bioreducible Phosphonoamidate Pro-drug Inhibitor of Enolase: Proof of Concept Study.

Glycolysis inhibition remains aspirational in cancer therapy. We recently described a promising phosphonate inhibitor of enolase for cancers harboring homozygous deletions of ENO1. Here, we describe the application of a nitroheterocycle phosphonoamidate pro-drug pair to capitalize on tumor hypoxia. This bioreducible prodrug exhibits greater-than 2-fold potency under hypoxic conditions compared to normoxia and exhibits robust stability in biological fluids. Our work provides strong in vitro proof-of-concept for using bioreduction as a pro-drug delivery strategy in the context of enolase inhibition.

The Rheb-TORC1 signaling axis functions as a developmental checkpoint.

In many eukaryotes, the small GTPase Rheb functions as a switch to toggle activity of TOR complex 1 (TORC1) between anabolism and catabolism, thus controlling lifespan, development and autophagy. Our CRISPR-generated, fluorescently tagged endogenous Caenorhabditis elegans RHEB-1 and DAF-15/Raptor are expressed ubiquitously and localize to lysosomes. LET-363/TOR and DAF-15/Raptor are required for development beyond the third larval stage (L3). We observed that deletion of RHEB-1 similarly conferred L3 arrest. Unexpectedly, robust RNAi-mediated depletion of TORC1 components caused arrest at stages prior to L3. Accordingly, conditional depletion of endogenous DAF-15/Raptor in the soma revealed that TORC1 is required at each stage of the life cycle to progress to the next stage. Reversal of DAF-15 depletion permits arrested animals to recover to continue development. Our results are consistent with TORC1 functioning as a developmental checkpoint that governs the decision of the animal to progress through development.