Clinton C. Mason

BCR-ABL1 compound mutations in tyrosine kinase inhibitor–resistant CML: frequency and clonal relationships

BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.

Podocyte detachment and reduced glomerular capillary endothelial fenestration promote kidney disease in type 2 diabetic nephropathy

Podocyte detachment and reduced endothelial cell fenestration and relationships between these features and the classic structural changes of diabetic nephropathy have not been described in patients with type 2 diabetes. Here we studied these relationships in 37 Pima Indians with type 2 diabetes of whom 11 had normal albuminuria, 16 had microalbuminuria, and 10 had macroalbuminuria. Biopsies from ten kidney donors (not Americans Indians) showed almost undetectable (0.03%) podocyte detachment and 43.5% endothelial cell fenestration. In patients with type 2 diabetes, by comparison, the mean percentage of podocyte detachment was significantly higher in macroalbuminuria (1.48%) than in normal albuminuria (0.41%) or microalbuminuria (0.37%). Podocyte detachment correlated significantly with podocyte number per glomerulus and albuminuria. The mean percentage of endothelial cell fenestration was significantly lower in macroalbuminuria (19.3%) than in normal (27.4%) or microalbuminuria (27.2%) and correlated significantly with glomerular basement membrane thickness, albuminuria, fractional mesangial area, and the glomerular filtration rate (iothalamate clearance). Podocyte detachment and diminished endothelial cell fenestration were not correlated, but were related to classic lesions of diabetic nephropathy. Thus, our findings confirm the important role these injuries play in the development and progression of kidney disease in type 2 diabetes, just as they do in type 1 diabetes. Whether podocyte detachment creates conduits for proteins to escape the glomerular circulation and reduced endothelial fenestration lowers glomerular hydraulic permeability requires further study.

Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia

Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen–deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.

Progression to Type 2 Diabetes Characterized by Moderate Then Rapid Glucose Increases

OBJECTIVE—The transition of an individual from normoglycemia to diabetes has generally been thought to involve either moderate or rapid changes in glucose over time, although few studies have analyzed these changes. We sought to determine whether a general pattern of glucose change exists in most individuals who become diabetic. RESEARCH DESIGN AND METHODS—We examined longitudinal data from Pima Indians who developed diabetes after several biennial examinations to characterize changes in 2-h plasma glucose. A distinct pattern of glucose change was apparent in the time course of most individuals, an initial linear trend followed by a steeper rise in glucose values. A model consisting of additive linear and exponential functions was hypothesized to account for this pattern and was tested for goodness of fit on 55 individuals who became diabetic after at least 10 previous examinations. RESULTS—The combined linear and exponential model provided a significantly better fit than linear or exponential models alone in 40 of the 55 cases (P < 10−38). Using this model, the timeframe over which glucose values rose suddenly was estimated, having a median time to onset of <4.5 years from the time at which the exponential effect had contributed a modest increase of 10 mg/dl to the initial linear trend. CONCLUSIONS—We conclude that there are two distinct processes affecting glucose levels in most individuals who progress to type 2 diabetes and that the rapid glucose rise identified in these people may be an important period for physiologic and preventive research.

Early Renal Function Decline in Type 2 Diabetes

BACKGROUND AND OBJECTIVES Early decline in GFR may reflect progressive kidney disease in type 1 diabetes, but its predictive value in type 2 diabetes is uncertain. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In this longitudinal study, GFR was measured serially over approximately 4.0 years in 195 Pima Indians with type 2 diabetes. Renal function decline (RFD) was defined during this initial period by an average GFR loss ≥3.3%/yr, as defined previously in type 1 diabetes. Subsequently, participants were followed for up to 17.8 years to ESRD onset, death, or December 31, 2010, whichever came first. RESULTS RFD prevalence during the initial period was 32% in 68 participants with normal baseline albuminuria (albumin/creatinine ratio [ACR] < 30 mg/g), 42% in 88 with microalbuminuria (ACR 30 to <300 mg/g), and 74% in 39 with macroalbuminuria (ACR ≥300 mg/g; P<0.001). The cumulative incidence of ESRD 10 years after the initial period was 41% in those with RFD and 15% in those without (P<0.001); 41 of the 49 ESRD cases (83.7%) occurred in participants who had or developed macroalbuminuria during the initial period. When adjusted for age, sex, diabetes duration, and hemoglobin A1c, the ESRD hazard rate was 4.78 times (95% confidence interval, 2.39-9.58) as high in those with RFD as in those without; further adjustment for albuminuria attenuated this association (hazard ratio, 1.79; 95% confidence interval, 0.82-3.91). CONCLUSIONS In type 2 diabetes, loss of GFR often occurs before the onset of macroalbuminuria, but a decline predictive of ESRD is strongly dependent on progression to macroalbuminuria.

The Association of ENPP1 K121Q with Diabetes Incidence Is Abolished by Lifestyle Modification in the Diabetes Prevention Program

CONTEXT Insulin resistance is an important feature of type 2 diabetes. Ectoenzyme nucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling, and a recent meta-analysis reported a nominal association between the Q allele in the K121Q (rs1044498) single nucleotide polymorphism in its gene ENPP1 and type 2 diabetes. OBJECTIVE AND INTERVENTION: We examined the impact of this polymorphism on diabetes incidence as well as insulin secretion and sensitivity at baseline and after treatment with a lifestyle intervention or metformin vs. placebo in the Diabetes Prevention Program (DPP). DESIGN, SETTING, PARTICIPANTS, AND OUTCOME: We genotyped ENPP1 K121Q in 3548 DPP participants and performed Cox regression analyses using genotype, intervention, and interactions as predictors of diabetes incidence. RESULTS Fasting glucose and glycated hemoglobin were higher in QQ homozygotes at baseline (P < 0.001 for both). There was a significant interaction between genotype at rs1044498 and intervention under the dominant model (P = 0.03). In analyses stratified by treatment arm, a positive association with diabetes incidence was found in Q allele carriers compared to KK homozygotes [hazard ratio (HR), 1.38; 95% confidence interval (CI), 1.08-1.76; P = 0.009] in the placebo arm (n = 996). Lifestyle modification eliminated this increased risk. These findings persisted after adjustment for body mass index and race/ethnicity. Association of ENPP1 K121Q genotype with diabetes incidence under the additive and recessive genetic models showed consistent trends [HR, 1.10 (95% CI, 0.99-1.23), P = 0.08; and HR, 1.16 (95% CI, 0.92-1.45), P = 0.20, respectively] but did not reach statistical significance. CONCLUSIONS ENPP1 K121Q is associated with increased diabetes incidence; the DPP lifestyle intervention eliminates this increased risk.

Arsenic Exposure and Incidence of Type 2 Diabetes in Southwestern American Indians

Association of urinary arsenic concentration with incident diabetes was examined in American Indians from Arizona who have a high prevalence of type 2 diabetes and were screened for diabetes between 1982 and 2007. The population resides where drinking water contains arsenic at concentrations above federally recommended limits. A total of 150 nondiabetic subjects aged ≥25 years who subsequently developed type 2 diabetes were matched by year of examination and sex to 150 controls who remained nondiabetic for ≥10 years. Total urinary arsenic concentration, adjusted for urinary creatinine level, ranged from 6.6 µg/L to 123.1 µg/L, and inorganic arsenic concentration ranged from 0.1 µg/L to 36.0 µg/L. In logistic regression models adjusted for age, sex, body mass index, and urinary creatinine level, the odds ratios for incident diabetes were 1.11 (95% confidence interval (CI): 0.79, 1.57) and 1.16 (95% CI: 0.89, 1.53) for a 2-fold increase in total arsenic and inorganic arsenic, respectively. Categorical analyses suggested a positive relationship between quartiles of inorganic arsenic and incident diabetes (P = 0.056); post-hoc comparison of quartiles 2-4 with quartile 1 revealed 2-fold higher odds of diabetes in the upper quartiles (OR = 2.14, 95% CI: 1.19, 3.85). Modestly elevated exposure to inorganic arsenic may predict type 2 diabetes in American Indians. Larger studies that include measures of speciated arsenic are required for confirmation.

Age-related mutations and chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a hematologic malignancy nearly confined to the elderly. Previous studies to determine incidence and prognostic significance of somatic mutations in CMML have relied on candidate gene sequencing, although an unbiased mutational search has not been conducted. As many of the genes commonly mutated in CMML were recently associated with age-related clonal hematopoiesis (ARCH) and aged hematopoiesis is characterized by a myelomonocytic differentiation bias, we hypothesized that CMML and aged hematopoiesis may be closely related. We initially established the somatic mutation landscape of CMML by whole exome sequencing followed by gene-targeted validation. Genes mutated in ⩾10% of patients were SRSF2, TET2, ASXL1, RUNX1, SETBP1, KRAS, EZH2, CBL and NRAS, as well as the novel CMML genes FAT4, ARIH1, DNAH2 and CSMD1. Most CMML patients (71%) had mutations in ⩾2 ARCH genes and 52% had ⩾7 mutations overall. Higher mutation burden was associated with shorter survival. Age-adjusted population incidence and reported ARCH mutation rates are consistent with a model in which clinical CMML ensues when a sufficient number of stochastically acquired age-related mutations has accumulated, suggesting that CMML represents the leukemic conversion of the myelomonocytic-lineage-biased aged hematopoietic system.

Bimodal distribution of RNA expression levels in human skeletal muscle tissue

BackgroundMany human diseases and phenotypes are related to RNA expression, levels of which are influenced by a wide spectrum of genetic and exposure-related factors. In a large genome-wide study of muscle tissue expression, we found that some genes exhibited a bimodal distribution of RNA expression, in contrast to what is usually assumed in studies of a single healthy tissue. As bimodality has classically been considered a hallmark of genetic control, we assessed the genome-wide prevalence, cause, and association of this phenomenon with diabetes-related phenotypes in skeletal muscle tissue from 225 healthy Pima Indians using exon array expression chips.ResultsTwo independent batches of microarrays were used for bimodal assessment and comparison. Of the 17,881 genes analyzed, eight (GSTM1, HLA-DRB1, ERAP2, HLA-DRB5, MAOA, ACTN3, NR4A2, and THNSL2) were found to have bimodal expression replicated in the separate batch groups, while 24 other genes had evidence of bimodality in only one group. Some bimodally expressed genes had modest associations with pre-diabetic phenotypes, of note ACTN3 with insulin resistance. Most of the other bimodal genes have been reported to be involved with various other diseases and characteristics. Association of expression with cis genetic variation in a subset of 149 individuals found all but one of the confirmed bimodal genes and nearly half of all potential ones to be highly significant expression quantitative trait loci (eQTL). The rare prevalence of these bimodally expressed genes found after controlling for batch effects was much lower than the prevalence reported in other studies. Additional validation in data from separate muscle expression studies confirmed the low prevalence of bimodality we observed.ConclusionsWe conclude that the prevalence of bimodal gene expression is quite rare in healthy muscle tissue (<0.2%), and is much lower than limited reports from other studies. The major cause of these clearly bimodal expression patterns in homogeneous tissue appears to be cis-polymorphisms, indicating that such bimodal genes are, for the most part, eQTL. The high frequency of disease associations reported with these genes gives hope that this unique feature may identify or actually be an underlying factor responsible for disease development.

Effects of Genetic Variants Previously Associated with Fasting Glucose and Insulin in the Diabetes Prevention Program

Common genetic variants have been recently associated with fasting glucose and insulin levels in white populations. Whether these associations replicate in pre-diabetes is not known. We extended these findings to the Diabetes Prevention Program, a clinical trial in which participants at high risk for diabetes were randomized to placebo, lifestyle modification or metformin for diabetes prevention. We genotyped previously reported polymorphisms (or their proxies) in/near G6PC2, MTNR1B, GCK, DGKB, GCKR, ADCY5, MADD, CRY2, ADRA2A, FADS1, PROX1, SLC2A2, GLIS3, C2CD4B, IGF1, and IRS1 in 3,548 Diabetes Prevention Program participants. We analyzed variants for association with baseline glycemic traits, incident diabetes and their interaction with response to metformin or lifestyle intervention. We replicated associations with fasting glucose at MTNR1B (P<0.001), G6PC2 (P = 0.002) and GCKR (P = 0.001). We noted impaired β-cell function in carriers of glucose-raising alleles at MTNR1B (P<0.001), and an increase in the insulinogenic index for the glucose-raising allele at G6PC2 (P<0.001). The association of MTNR1B with fasting glucose and impaired β-cell function persisted at 1 year despite adjustment for the baseline trait, indicating a sustained deleterious effect at this locus. We also replicated the association of MADD with fasting proinsulin levels (P<0.001). We detected no significant impact of these variants on diabetes incidence or interaction with preventive interventions. The association of several polymorphisms with quantitative glycemic traits is replicated in a cohort of high-risk persons. These variants do not have a detectable impact on diabetes incidence or response to metformin or lifestyle modification in the Diabetes Prevention Program.