Brian J. Skaggs

Sequential ABL kinase inhibitor therapy selects for compound drug-resistant BCR-ABL mutations with altered oncogenic potency

Molecularly targeted kinase inhibitor cancer therapies are currently administered sequentially rather than simultaneously. We addressed the potential long-term impact of this strategy in patients with chronic myelogenous leukemia (CML), which is driven by the fusion oncogene BCR-ABL. Analysis of BCR-ABL genotypes in CML patients who relapsed after sequential treatment with the ABL inhibitors imatinib and dasatinib revealed evolving resistant BCR-ABL kinase domain mutations in all cases. Twelve patients relapsed with the pan-resistant T315I mutation, whereas 6 patients developed novel BCR-ABL mutations predicted to retain sensitivity to imatinib based on in vitro studies. Three of these patients were retreated with imatinib (or the chemically related compound nilotinib) and responded; however, selection for compound mutants (2 or 3 BCR-ABL mutations in the same molecule) can substantially limit the potential effectiveness of retreating patients with inhibitors that have previously failed. Furthermore, drug-resistant mutations, when compounded, can increase oncogenic potency relative to the component mutants in transformation assays. The Aurora kinase inhibitor VX-680, currently under clinical evaluation based on its activity against the T315I mutation, is also effective against the other commonly detected dasatinib-resistant mutation in our analysis, V299L. Our findings demonstrate the potential hazards of sequential kinase inhibitor therapy and suggest a role for a combination of ABL kinase inhibitors, perhaps including VX-680, to prevent the outgrowth of cells harboring drug-resistant BCR-ABL mutations.

Dysfunctional Proinflammatory High-Density Lipoproteins Confer Increased Risk of Atherosclerosis in Women With Systemic Lupus Erythematosus

OBJECTIVE Women with systemic lupus erythematosus (SLE) have an increased risk of atherosclerosis. Identification of at-risk patients and the etiology underlying atherosclerosis in SLE remain elusive. The antioxidant capacity of normal high-density lipoproteins (HDLs) is lost during inflammation, and these dysfunctional HDLs might predispose individuals to atherosclerosis. The aim of this study was to determine whether dysfunctional proinflammatory HDL (piHDL) is associated with subclinical atherosclerosis in SLE. METHODS Carotid artery ultrasound was performed in 276 women with SLE to identify carotid plaques and measure intima-media thickness (IMT). The antioxidant function of HDL was measured as the change in oxidation of low-density lipoprotein after the addition of HDL cholesterol. Two antiinflammatory HDL components, paraoxonase 1 and apolipoprotein A-I, were also measured. RESULTS Among the SLE patients, 48.2% were determined to have piHDL on carotid ultrasound, while 86.7% of patients with plaque had piHDL compared with 40.7% of those without plaque (P<0.001). Patients with piHDL also had a higher IMT (P<0.001). After multivariate analysis, the only factors found to be significantly associated with plaque were the presence of piHDL (odds ratio [OR] 16.1, P<0.001), older age (OR 1.2, P<0.001), hypertension (OR 3.0, P=0.04), dyslipidemia (OR 3.4, P=0.04), and mixed racial background (OR 8.3, P=0.04). Factors associated with IMT measurements in the highest quartile were the presence of piHDL (OR 2.5, P=0.02), older age (OR 1.1, P<0.001), a higher body mass index (OR 1.07, P=0.04), a cumulative lifetime prednisone dose>or=20 gm (OR 2.9, P=0.04), and African American race (OR 8.3, P=0.001). CONCLUSION Dysfunctional piHDL greatly increases the risk of developing subclinical atherosclerosis in SLE. The presence of piHDL was associated with an increased prevalence of carotid plaque and with a higher IMT. Therefore, determination of piHDL may help identify patients at risk for atherosclerosis.

Accelerated atherosclerosis in patients with SLE—mechanisms and management

Rapid-onset cardiovascular disease (CVD) is a major concern for many patients with systemic lupus erythematosus (SLE). Cardiovascular events occur more frequently and with earlier onset in patients with SLE compared with healthy individuals. Traditional risk factors, such as altered lipid levels, aging and smoking, do not fully explain this increased risk of CVD, strongly suggesting that autoimmunity contributes to accelerated atherosclerosis. Altered immune system function is recognized as the primary contributor to both the initiation and progression of atherosclerosis. Multiple manifestations of autoimmunity, including changes in cytokine levels and innate immune responses, autoantibodies, adipokines, dysfunctional lipids, and oxidative stress, could heighten atherosclerotic risk. In addition, multiple SLE therapeutics seem to affect the development and progression of atherosclerosis both positively and negatively. SLE-specific cardiovascular risk factors are beginning to be discovered by several groups, and development of a comprehensive, clinically feasible biomarker panel could be invaluable for identification and treatment of patients at risk of developing accelerated atherosclerosis. Here, we discuss the epidemiology of CVD in SLE and the implications of immune system dysfunction on the development and progression, monitoring and treatment of atherosclerosis in individuals with this disease.

Phosphorylation of the ATP-binding loop directs oncogenicity of drug-resistant BCR-ABL mutants

The success of targeting kinases in cancer with small molecule inhibitors has been tempered by the emergence of drug-resistant kinase domain mutations. In patients with chronic myeloid leukemia treated with ABL inhibitors, BCR-ABL kinase domain mutations are the principal mechanism of relapse. Certain mutations are occasionally detected before treatment, suggesting increased fitness relative to wild-type p210 BCR-ABL. We evaluated the oncogenicity of eight kinase inhibitor-resistant BCR-ABL mutants and found a spectrum of potencies greater or less than p210. Although most fitness alterations correlate with changes in kinase activity, this is not the case with the T315I BCR-ABL mutation that confers clinical resistance to all currently approved ABL kinase inhibitors. Through global phosphoproteome analysis, we identified a unique phosphosubstrate signature associated with each drug-resistant allele, including a shift in phosphorylation of two tyrosines (Tyr253 and Tyr257) in the ATP binding loop (P-loop) of BCR-ABL when Thr315 is Ile or Ala. Mutational analysis of these tyrosines in the context of Thr315 mutations demonstrates that the identity of the gatekeeper residue impacts oncogenicity by altered P-loop phosphorylation. Therefore, mutations that confer clinical resistance to kinase inhibitors can substantially alter kinase function and confer novel biological properties that may impact disease progression.

High plasma leptin levels confer increased risk of atherosclerosis in women with systemic lupus erythematosus, and are associated with inflammatory oxidised lipids

Background Patients with systemic lupus erythematosus (SLE) are at increased risk of atherosclerosis, even after accounting for traditional risk factors. High levels of leptin and low levels of adiponectin are associated with both atherosclerosis and immunomodulatory functions in the general population. Objective To examine the association between these adipokines and subclinical atherosclerosis in SLE, and also with other known inflammatory biomarkers of atherosclerosis. Methods Carotid ultrasonography was performed in 250 women with SLE and 122 controls. Plasma leptin and adiponectin levels were measured. Lipoprotein a (Lp(a)), oxidised phospholipids on apoB100 (OxPL/apoB100), paraoxonase, apoA-1 and inflammatory high-density lipoprotein (HDL) function were also assessed. Results Leptin levels were significantly higher in patients with SLE than in controls (23.7±28.0 vs 13.3±12.9 ng/ml, p<0.001). Leptin was also higher in the 43 patients with SLE with plaque than without plaque (36.4±32.3 vs 20.9±26.4 ng/ml, p=0.002). After multivariate analysis, the only significant factors associated with plaque in SLE were leptin levels in the highest quartile (≥29.5 ng/ml) (OR=2.8, p=0.03), proinflammatory HDL (piHDL) (OR=12.8, p<0.001), age (OR=1.1, p<0.001), tobacco use (OR=7.7, p=0.03) and hypertension (OR=3.0, p=0.01). Adiponectin levels were not significantly associated with plaque in our cohort. A significant correlation between leptin and piHDL function (p<0.001), Lp(a) (p=0.01) and OxPL/apoB100 (p=0.02) was also present. Conclusions High leptin levels greatly increase the risk of subclinical atherosclerosis in SLE, and are also associated with an increase in inflammatory biomarkers of atherosclerosis such as piHDL, Lp(a) and OxPL/apoB100. High leptin levels may help to identify patients with SLE at risk of atherosclerosis.

CD8+ Tregs in lupus, autoimmunity, and beyond.

While CD4(+)CD25(high) regulatory T cells (Tregs) have garnered much attention for their role in the maintenance of immune homeostasis, recent findings have shown that subsets of CD8(+) T cells (CD8(+) Tregs) display immunoregulatory functions as well. Both CD4(+) Tregs and CD8(+) Tregs appear impaired in number and/or function in several autoimmune diseases and in experimental animal models of autoimmunity, suggesting the possibility of immunotherapeutic targeting of these cells for improved management of autoimmune conditions. Our group has developed a strategy to induce CD8(+) Tregs in autoimmune mice through the use of a tolerogenic self-peptide, and new information has been gained on the phenotype, function and role of induced CD8(+) Tregs in autoimmunity. Here we present an overview of the role and mechanisms of action of CD8(+) Tregs in autoimmunity, with a special focus on lupus. We also discuss the potential role of CD8(+) Tregs in other diseases, including chronic infection and cancer.

Altered lipoprotein metabolism in chronic inflammatory states: proinflammatory high-density lipoprotein and accelerated atherosclerosis in systemic lupus erythematosus and rheumatoid arthritis.

In this review, the authors discuss the formation and structure of high-density lipoproteins (HDLs) and how those particles are altered in inflammatory or stress states to lose their capacity for reverse cholesterol transport and for antioxidant activity. In addition, abnormal HDLs can become proinflammatory (piHDLs) and actually contribute to oxidative damage. The assay by which piHDLs are identified involves studying the ability of test HDLs to prevent oxidation of low-density lipoproteins. Finally, the authors discuss the potential role of piHDLs (found in some 45% of patients with systemic lupus erythematosus and 20% of patients with rheumatoid arthritis) in the accelerated atherosclerosis associated with some chronic rheumatic diseases.

Low Physical Activity is Associated with Proinflammatory High Density Lipoprotein and Increased Subclinical Atherosclerosis in Women with Systemic Lupus Erythematosus

To investigate the association between physical activity, functional activity of high‐density lipoprotein (HDL), and subclinical cardiovascular disease in patients with systemic lupus erythematosus (SLE).

Global Phosphoproteomics Reveals Crosstalk Between Bcr-Abl and Negative Feedback Mechanisms Controlling Src Signaling

Negative feedback fails to limit Src family kinase activity in the presence of Bcr-Abl, an oncoprotein that drives leukemia. When Negative Feedback Fails Bcr-Abl is a fusion protein with tyrosine kinase activity that causes some forms of leukemia. Bcr-Abl activates Src family tyrosine kinases (SFKs), but resistance to drugs, such as dasatinib and imatinib, that target Bcr-Abl and SFKs limits their clinical usefulness. With global tyrosine phosphoproteomic analysis in a murine leukemia cell line model, Rubbi et al. identified several negative feedback mechanisms that limited SFK activity and showed that their effectiveness was blunted by Bcr-Abl. Sensitivity of human leukemia cell lines to imatinib correlated with the amount of negative feedback signaling to SFKs. By exploring the mechanisms by which Bcr-Abl overpowered the negative feedback, the authors identified potential therapeutic targets for treating leukemias resistant to Bcr-Abl and SFK inhibitors or that may be combined with these tyrosine kinase inhibitors to prevent the development of resistance. In subtypes and late stages of leukemias driven by the tyrosine kinase fusion protein Bcr-Abl, signaling by the Src family kinases (SFKs) critically contributes to the leukemic phenotype. We performed global tyrosine phosphoprofiling by quantitative mass spectrometry of Bcr-Abl–transformed cells in which the activities of the SFKs were perturbed to build a detailed context-dependent network of cancer signaling. Perturbation of the SFKs Lyn and Hck with genetics or inhibitors revealed Bcr-Abl downstream phosphorylation events either mediated by or independent of SFKs. We identified multiple negative feedback mechanisms within the network of signaling events affected by Bcr-Abl and SFKs and found that Bcr-Abl attenuated these inhibitory mechanisms. The C-terminal Src kinase (Csk)–binding protein Pag1 (also known as Cbp) and the tyrosine phosphatase Ptpn18 both mediated negative feedback to SFKs. We observed Bcr-Abl–mediated phosphorylation of the phosphatase Shp2 (Ptpn11), and this may contribute to the suppression of these negative feedback mechanisms to promote Bcr-Abl–activated SFK signaling. Csk and a kinase-deficient Csk mutant both produced similar globally repressive signaling consequences, suggesting a critical role for the adaptor protein function of Csk in its inhibition of Bcr-Abl and SFK signaling. The identified Bcr-Abl–activated SFK regulatory mechanisms are candidates for dysregulation during leukemia progression and acquisition of SFK-mediated drug resistance.

A Panel of Biomarkers Is Associated With Increased Risk of the Presence and Progression of Atherosclerosis in Women With Systemic Lupus Erythematosus

An increased frequency of atherosclerosis (ATH) in systemic lupus erythematosus (SLE) is well‐documented but not fully explained by the presence of traditional cardiac risk factors. Several nontraditional biomarkers, including proinflammatory high‐density lipoprotein (piHDL) and leptin, have been individually associated with subclinical ATH in SLE. The aim of this study was to examine whether these and other biomarkers can be combined into a risk profile, the Predictors of Risk for Elevated Flares, Damage Progression, and Increased Cardiovascular Disease in Patients with SLE (PREDICTS), that could be used to better predict future progression of ATH.