Karen S. Anderson

Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors

The mechanism of inhibition of HIV-1 reverse transcriptase by three nonnucleoside inhibitors is described. Nevirapine, O-TIBO, and CI-TIBO each bind to a hydrophobic pocket in the enzyme-DNA complex close to the active site catalytic residues. Pre-steady-state kinetic analysis was used to establish the mechanism of inhibition by these noncompetitive inhibitors. Analysis of the pre-steady-state burst of DNA polymerization indicated that inhibitors blocked the chemical reaction, but did not interfere with nucleotide binding or the nucleotide-induced conformational change. Rather, in the presence of saturating concentrations of the inhibitors, the nucleoside triphosphate bound tightly (Kd, 100 nM), but nonproductively. The data suggest that an inhibitor combining the functionalities of a nonnucleoside inhibitor and a nucleotide analog could bind very tightly and specifically to reverse transcriptase and could be effective in the treatment of AIDS.

The JAK2/STAT3 signaling pathway is required for growth of CD44+CD24– stem cell–like breast cancer cells in human tumors

Intratumor heterogeneity is a major clinical problem because tumor cell subtypes display variable sensitivity to therapeutics and may play different roles in progression. We previously characterized 2 cell populations in human breast tumors with distinct properties: CD44+CD24- cells that have stem cell-like characteristics, and CD44-CD24+ cells that resemble more differentiated breast cancer cells. Here we identified 15 genes required for cell growth or proliferation in CD44+CD24- human breast cancer cells in a large-scale loss-of-function screen and found that inhibition of several of these (IL6, PTGIS, HAS1, CXCL3, and PFKFB3) reduced Stat3 activation. We found that the IL-6/JAK2/Stat3 pathway was preferentially active in CD44+CD24- breast cancer cells compared with other tumor cell types, and inhibition of JAK2 decreased their number and blocked growth of xenografts. Our results highlight the differences between distinct breast cancer cell types and identify targets such as JAK2 and Stat3 that may lead to more specific and effective breast cancer therapies.

Vaccination of Cancer Patients Against Telomerase Induces Functional Antitumor CD8+ T Lymphocytes

Purpose: High-level expression of the telomerase reverse transcriptase (hTERT) in >85% of human cancers, in contrast with its restricted expression in normal adult tissues, points to hTERT as a broadly applicable molecular target for anticancer immunotherapy. CTLs recognize peptides derived from hTERT and kill hTERT+ tumor cells of multiple histologies in vitro. Moreover, because survival of hTERT+ tumor cells requires functionally active telomerase, hTERT mutation or loss as a means of escape may be incompatible with sustained tumor growth. Experimental Design: A Phase I clinical trial was performed to evaluate the clinical and immunological impact of vaccinating advanced cancer patients with the HLA-A2-restricted hTERT I540 peptide presented with keyhole limpet hemocyanin by ex vivo generated autologous dendritic cells. Results: As measured by peptide/MHC tetramer, enzyme-linked immunospot, and cytotoxicity assays, hTERT-specific T lymphocytes were induced in 4 of 7 patients with advanced breast or prostate carcinoma after vaccination with dendritic cells pulsed with hTERT peptide. Tetramer-guided high-speed sorting and polyclonal expansion achieved highly enriched populations of hTERT-specific cells that killed tumor cells in an MHC- restricted fashion. Despite concerns of telomerase activity in rare normal cells, no significant toxicity was observed. Partial tumor regression in 1 patient was associated with the induction of CD8+ tumor infiltrating lymphocytes. Conclusions: These results demonstrate the immunological feasibility of vaccinating patients against telomerase and provide rationale for targeting self-antigens with critical roles in oncogenesis.

Dose-response curve slope sets class-specific limits on inhibitory potential of anti-HIV drugs

Highly active antiretroviral therapy (HAART) can control HIV-1 replication, but suboptimal treatment allows for the evolution of resistance and rebound viremia. A comparative measure of antiviral activity under clinically relevant conditions would guide drug development and the selection of regimens that maximally suppress replication. Here we show that current measures of antiviral activity, including IC50 and inhibitory quotient, neglect a key dimension, the dose-response curve slope. Using infectivity assays with wide dynamic range, we show that this slope has noteworthy effects on antiviral activity. Slope values are class specific for antiviral drugs and define intrinsic limitations on antiviral activity for some classes. Nucleoside reverse transcriptase inhibitors and integrase inhibitors have slopes of ∼1, characteristic of noncooperative reactions, whereas non-nucleoside reverse transcriptase inhibitors, protease inhibitors and fusion inhibitors unexpectedly show slopes >1. Instantaneous inhibitory potential (IIP), the log reduction in single-round infectivity at clinical drug concentrations, is strongly influenced by slope and varies by >8 logs for anti-HIV drugs. IIP provides a more accurate measure of antiviral activity and in general correlates with clinical outcomes. Only agents with slopes >1 achieve high-level inhibition of single-round infectivity, a finding with profound implications for drug and vaccine development.

Oncologists' reluctance to accrue patients onto clinical trials: an Illinois Cancer Center study.

Clinical trials are recognized as the standard of care for the cancer patient, and the randomized, controlled trial represents the most definitive method to determine the effectiveness or ineffectiveness of a cancer treatment. However, less than 3% of all eligible patients enter a clinical trial. Of the 437 physician members of the Illinois Cancer Center (ICC), 244 responded to a survey designed to determine factors that present a significant barrier to entering patients on clinical trials. Rigid protocol design was the primary deterrent to accrual, especially for medical oncologists. Surgeons, radiation oncologists, and medical oncologists differed with respect to several factors, including willingness to seek a clinical trial, tendency to treat patients off study, quality-of-life issues, and the belief that trials were too excessive in time commitment (P less than .05). Compared with hospital-based physicians, community oncologists had fewer patients on trial, were more likely to enter patients on the basis of age, and were more concerned about aspects of informed consent and the financial burden of a trial (P less than .01). One third of the physicians never pursued a clinical trial because of conflict with the priorities of individual care and excessive follow-up time. Fourteen percent indicated that they discouraged patients from participating in a clinical trial due to the risk of a patient receiving a placebo and patient follow-up requirements (P less than .05). Subgroups of physicians differ in their reluctance to accrue patients, and there are clusters of beliefs expressed by physicians concerning their clinical trial activity. Current conduct of clinical trials needs to be reassessed, and intervention studies are required to determine the best methodology to alter physician reluctance to pursue clinical trials.

Protein Microarray Signature of Autoantibody Biomarkers for the Early Detection of Breast Cancer

Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a prescreen was performed to eliminate uninformative antigens. Sera from stage I-III IBC (n = 53) and healthy women (n = 53) were screened for AAb to all 4988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p < 0.05) and if the number of cases with signals above the 95th percentile of controls was significant (p < 0.05). These 761 antigens were screened using an independent set of IBC sera (n = 51) and sera from women with benign breast disease (BBD) (n = 39). From these, 119 antigens had a partial area under the ROC curve (p < 0.05), with sensitivities ranging from 9-40% at >91% specificity. Twenty-eight of these antigens were confirmed using an independent serum cohort (n = 51 cases/38 controls, p < 0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC = 0.756). These are potential biomarkers for the early detection of breast cancer.

Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum; may exist in greater concentrations than their cognate antigens; and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.

Insights into the Molecular Mechanism of Mitochondrial Toxicity by AIDS Drugs

Several of the nucleoside analogs used in the treatment of AIDS exhibit a delayed clinical toxicity limiting their usefulness. The toxicity of nucleoside analogs may be related to their effects on the human mitochondrial DNA polymerase (Pol γ), the polymerase responsible for mitochondrial DNA replication. Among the AIDS drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (2′,3′-dideoxycytidine (ddC)) and Lamivudine (β-d-(+)-2′,3′-dideoxy-3′-thiacytidine ((−)3TC])). (−)3TC is the only analog containing an unnaturall(−) nucleoside configuration and is well tolerated by patients even after long term administration. In cell culture (−)3TC is less toxic than its d(+) isomer, (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. We have investigated the mechanistic basis for the differential toxicity of these three cytosine analogs by comparing the effects of dideoxy-CTP), (+)3TC-triphosphate (TP), and (−)3TC-TP on the polymerase and exonuclease activities of recombinant human Pol γ. This analysis reveals that Pol γ incorporates (−)3TC-triphosphate 16-fold less efficiently than the corresponding (+)isomer and 1140-fold less efficiently than dideoxy-CTP, showing a good correlation between incorporation rate and toxicity. The rates of excision of the incorporated analogs from the chain-terminated 3′-end of the DNA primer by the 3′-5′-exonuclease activity of Pol γ were similar (0.01 s− 1) for both 3TC analogs. In marked contrast, the rate of exonuclease removal of a ddC chain-terminated DNA occurs at least 2 orders of magnitude slower, suggesting that the failure of the exonuclease to remove ddC may play a major role in its greater toxicity. This study demonstrates that direct analysis of the mitochondrial DNA polymerase structure/function relationships may provide valuable insights leading to the design of less toxic inhibitors.

A role for calnexin (IP90) in the assembly of class II MHC molecules.

Major histocompatibility complex (MHC) class II antigens consist of alpha and beta chains that associate intracellularly with the invariant (I) chain. The HLA‐DR alpha beta I complex assembles in the endoplasmic reticulum (ER) into a nonameric structure via progressive addition of three alpha beta dimers to a core invariant chain trimer. We have examined intracellular association of alpha beta I complexes with the resident ER protein calnexin. Calnexin associates rapidly (within 3 min) with newly synthesized alpha, beta and I chains, and remains associated with the assembling alpha beta I complex until the final alpha beta dimer is added, forming the complete nonamer. Dissociation of calnexin parallels egress of alpha beta I from the ER. These results suggest that calnexin retains and stabilizes both free class II subunits and partially assembled class II‐I chain complexes until assembly of the nonamer is complete.

Oral Human Papillomavirus (HPV) Infection in HPV-Positive Patients With Oropharyngeal Cancer and Their Partners

PURPOSE To better understand oral human papillomavirus (HPV) infection and cancer risk among long-term sexual partners of patients with HPV-positive oropharyngeal cancer (HPV-OPC). PATIENTS AND METHODS An oral rinse sample, risk factor survey, cancer history, and oral examination (partners only) were collected from patients with HPV-OPC and their partners. Oral rinse samples were evaluated for 36 types of HPV DNA using PGMY 09/11 primers and line-blot hybridization and HPV16 copy number using quantitative polymerase chain reaction. Oral HPV prevalence was compared with infection among those age 45 to 65 years using National Health and Nutrition Examination Survey (NHANES) 2009-2010. RESULTS A total of 164 patients with HPV-OPC and 93 of their partners were enrolled. Patients were primarily men (90%), were never-smokers (51%), and had performed oral sex (97%), with a median age of 56 years; they had a high prevalence of oncogenic oral HPV DNA (61%) and oral HPV16 DNA (54%) at enrollment. Female partners had comparable oncogenic oral HPV prevalence compared with members of the general population of the same age (1.2% v 1.3%). Among the six male partners, no oncogenic oral HPV infections were detected. No precancers or cancers were identified during partner oral cancer screening examinations. However, a history of cervical disease was reported by nine partners (10.3%) and two female patients (11.8%), and three patients (2.0%) reported a previous partner who developed invasive cervical cancer. CONCLUSION Oral HPV16 DNA is commonly detected among patients with HPV-OPC at diagnosis, but not among their partners. Partners of patients with HPV-OPC do not seem to have elevated oral HPV infection compared with the general population.