Ming Poi

Regulatory mechanisms of tumor suppressor P16(INK4A) and their relevance to cancer.

P16(INK4A) (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly because of the prevalence of genetic inactivation of the p16(INK4A) (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that an elevated level of expression (upregulation) of P16 is involved in cellular senescence, aging, and cancer progression, indicating that the regulation of P16 is critical for its function. Here, we discuss the regulatory mechanisms of P16 function at the DNA level, the transcription level, and the posttranscriptional level, as well as their implications for the structure-function relationship of P16 and for human cancers.

Somatic INK4a‐ARF locus mutations: A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck

The INK4a‐ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor‐suppressor genes. The p16INK4a (p16) tumor‐suppressor gene product is a negative regulator of cyclin‐dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14ARF tumor‐suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G1 arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0–10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14ARF genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single‐stranded conformational polymorphism (SSCP) analysis termed “cold” SSCP. Exons 1α, 1β, and 2 of INK4a/ARF were amplified using intron‐based primers or a combination of intron‐ and exon‐based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1α. No mutations were found in exon 1β. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14ARF proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin‐dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild‐type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29‐ to 38‐fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14ARF proteins, seems to be a prevalent event in the development of SCCHN. Mol. Carcinog. 30:26–36, 2001.

Common CYP2D6 polymorphisms affecting alternative splicing and transcription: long-range haplotypes with two regulatory variants modulate CYP2D6 activity

Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 25% of clinically used drugs. Genetic polymorphisms cause substantial variation in CYP2D6 activity and serve as biomarkers guiding drug therapy. However, genotype-phenotype relationships remain ambiguous except for poor metabolizers carrying null alleles, suggesting the presence of yet unknown genetic variants. Searching for regulatory CYP2D6 polymorphisms, we find that a SNP defining the CYP2D6*2 allele, rs16947 [R296C, 17-60% minor allele frequency (MAF)], previously thought to convey normal activity, alters exon 6 splicing, thereby reducing CYP2D6 expression at least 2-fold. In addition, two completely linked SNPs (rs5758550/rs133333, MAF 13-42%) increase CYP2D6 transcription more than 2-fold, located in a distant downstream enhancer region (>100 kb) that interacts with the CYP2D6 promoter. In high linkage disequilibrium (LD) with each other, rs16947 and the enhancer SNPs form haplotypes that affect CYP2D6 enzyme activity in vivo. In a pediatric cohort of 164 individuals, rs16947 alone (minor haplotype frequency 28%) was associated with reduced CYP2D6 metabolic activity (measured as dextromethorphan/metabolite ratios), whereas rs5758550/rs133333 alone (frequency 3%) resulted in increased CYP2D6 activity, while haplotypes containing both rs16947 and rs5758550/rs133333 were similar to the wild-type. Other alleles used in biomarker panels carrying these variants such as CYP2D6*41 require re-evaluation of independent effects on CYP2D6 activity. The occurrence of two regulatory variants of high frequency and in high LD, residing on a long haplotype, highlights the importance of gene architecture, likely shaped by evolutionary selection pressures, in determining activity of encoded proteins.

Flavopiridol can be safely administered using a pharmacologically derived schedule and demonstrates activity in relapsed and refractory non-Hodgkin's lymphoma

Flavopiridol is a broad cyclin‐dependent kinase inhibitor (CDKI) that induces apoptosis of malignant lymphocytes in vitro and in murine lymphoma models. We conducted a Phase I dose‐escalation study to determine the maximum tolerated dose (MTD) for single‐agent flavopiridol administered on a pharmacokinetically derived hybrid dosing schedule to patients with relapsed and refractory non‐Hodgkins lymphoma. Dose was escalated independently in one of four cohorts: indolent B‐cell (Cohort 1), mantle cell (Cohort 2), intermediate‐grade B‐cell including transformed lymphoma (Cohort 3), and T‐/NK‐cell excluding primary cutaneous disease (Cohort 4). Forty‐six patients were accrued. Grade 3 or 4 leukopenia was observed in the majority of patients (60%), but infection was infrequent. Common nonhematologic toxicities included diarrhea and fatigue. Biochemical tumor lysis was observed in only two patients, and no patients required hemodialysis for its management. Dose escalation was completed in two cohorts (indolent and aggressive B‐cell). Dose‐limiting toxicities were not observed, and the MTD was not reached in either cohort at the highest dose tested (50 mg/m2 bolus + 50 mg/m2 continuous infusion weekly for 4 consecutive weeks of a 6‐week cycle). Clinical benefit was observed in 26% of 43 patients evaluable for response, including 14% with partial responses (two mantle cells, three indolent B‐cells, and one diffuse large B‐cell). The single‐agent activity of this first‐generation CDKI suggests that other agents in this class merit further study in lymphoid malignancies, both alone and in combination. Am. J. Hematol. 89:19–24, 2014.

A Pharmacokinetic/Pharmacodynamic Model of Tumor Lysis Syndrome in Chronic Lymphocytic Leukemia Patients Treated with Flavopiridol

Purpose: Flavopiridol, the first clinically evaluated cyclin-dependent kinase inhibitor, shows activity in patients with refractory chronic lymphocytic leukemia, but prevalent and unpredictable tumor lysis syndrome (TLS) presents a major barrier to its broad clinical use. The purpose of this study was to investigate the relationships between pretreatment risk factors, drug pharmacokinetics, and TLS. Experimental Design: A population pharmacokinetic/pharmacodynamic model linking drug exposure and TLS was developed. Plasma data of flavopiridol and its glucuronide metabolite (flavo-G) were obtained from 111 patients treated in early-phase trials with frequent sampling following initial and/or escalated doses. TLS grading was modeled with logistic regression as a pharmacodynamic endpoint. Demographics, baseline disease status, and blood chemistry variables were evaluated as covariates. Results: Gender was the most significant pharmacokinetic covariate, with females displaying higher flavo-G exposure than males. Glucuronide metabolite exposure was predictive of TLS occurrence, and bulky lymphadenopathy was identified as a significant covariate on TLS probability. The estimated probability of TLS occurrence in patients with baseline bulky lymphadenopathy less than 10 cm or 10 cm or more during the first 2 treatments was 0.111 (SE% 13.0%) and 0.265 (SE% 17.9%), respectively, when flavo-G area under the plasma concentration versus time curve was at its median value in whole-patient group. Conclusions: This is the first population pharmacokinetic/pharmacodynamic model of TLS. Further work is needed to explore potential mechanisms and to determine whether the associations between TLS, gender, and glucuronide metabolites are relevant in patients with chronic lymphocytic leukemia treated with other cyclin-dependent kinase inhibitors. Clin Cancer Res; 19(5); 1269–80. ©2012 AACR.

The combination of milatuzumab, a humanized anti-CD74 antibody, and veltuzumab, a humanized anti-CD20 antibody, demonstrates activity in patients with relapsed and refractory B-cell non-Hodgkin lymphoma

As a result of the anti‐tumour activity observed in vitro and in vivo with combined anti‐CD20 and anti‐CD74 antibodies, we initiated a phase I/II trial of veltuzumab and milatuzumab in patients with relapsed or refractory B‐cell non‐Hodgkin lymphoma (NHL). Patients received an induction of veltuzumab 200 mg/m2 weekly combined with escalating doses of milatuzumab at 8, 16 and 20 mg/kg weekly for 4 weeks. Patients without disease progression could receive an extended induction with treatment on weeks 12, 20, 28 and 36. A total of 35 patients enrolled on the study. Median age was 63 years, median number of prior therapies was 3, and 63% of patients were rituximab refractory. No dose‐limiting toxicities were observed in the phase I study. Related grade 3–4 toxicities included lymphopenia, leucopenia, neutropenia, anaemia, infusion reactions, hyperglycaemia, fatigue and atrial tachycardia. Median weeks of therapy was 12 and 29% of patients completed all 36 weeks of therapy. The overall response rate was 24%, median duration of response was 12 months, and responses were observed at all dose levels and in 50% of patients refractory to rituximab. Combination therapy with veltuzumab and milatuzumab demonstrated activity in a population of heavily pre‐treated patients with relapsed or refractory indolent NHL.

A Low-barrier Hydrogen Bond Between Histidine of Secreted Phospholipase A2 and a Transition State Analog Inhibitor

This work describes in-depth NMR characterization of a unique low-barrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A(2) (sPLA(2), from bee venom and bovine pancreas) and a transition-state analog inhibitor HK32. A downfield proton NMR resonance, at 17-18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48. These results led to a hypothesis that the downfield resonance represents the proton (H(epsilon 2) of H48) involved in the H-bonding between D99 and H48, in analogy with serine proteases. However, this was shown not to be the case by use of the bovine enzyme labeled with specific [15N(epsilon 2)]His. Instead, the downfield resonance arises from H(delta1) of H48, which forms a hydrogen bond with a non-bridging phosphonate oxygen of the inhibitor. Further studies showed that this proton displays a fractionation factor of 0.62(+/-0.06), and an exchange rate protection factor of >100 at 285 K and >40 at 298 K, which are characteristic of a LBHB. The pK(a) of the imidazole ring of H48 was shown to be shifted from 5.7 for the free enzyme to an apparent value of 9.0 in the presence of the inhibitor. These properties are very similar to those of the Asp em leader His LBHBs in serine proteases. Possible structural bases and functional consequences for the different locations of the LBHB between these two types of enzymes are discussed. The results also underscore the importance of using specific isotope labeling, rather than extrapolation of NMR results from other enzyme systems, to assign the downfield proton resonance to a specific hydrogen bond. Although our studies did not permit the strength of the LBHB to be accurately measured, the data do not provide support for an unusually strong hydrogen bond strength (i.e. >10 kcal/mol).

A dose escalation feasibility study of lenalidomide for treatment of symptomatic, relapsed chronic lymphocytic leukemia.

Adequate dosing of lenalidomide in Chronic Lymphocytic Leukemia (CLL) remains unclear. This study determined maximum tolerated dose (MTD) in relapsed CLL patients (Cohort A) and patients achieving a partial response (PR) or better to recent therapy (Cohort B). Thirty-seven patients were enrolled. MTD was 2.5mg followed by 5.0mg continuous. In Cohort A, tumor flare grade 1-2 occurred in 15 patients (50%) and grade 3 in 1 patient (3%). Cohort A had 19 of 23 evaluable (83%) patients, 4 PR (17%) and 15 (65%) stable disease (SD), Cohort B had 6 of 7 patients (86%) with SD. Despite overall response rate not being high, many patients remained on therapy several months with SD.

Erlotinib in African Americans With Advanced Non–Small Cell Lung Cancer: A Prospective Randomized Study With Genetic and Pharmacokinetic Analyses

Prospective studies on epidermal growth factor receptor (EGFR) inhibitors in African Americans with non–small cell lung cancer (NSCLC) have not previously been performed. In this phase II randomized study, 55 African Americans with NSCLC received 150 mg/day erlotinib or a body weight–adjusted dose with subsequent escalations to the maximum‐allowable dose, 200 mg/day, to achieve rash. Erlotinib and OSI‐420 exposures were lower than those observed in previous studies, consistent with CYP3A pharmacogenetics implying higher metabolic activity. Tumor genetics showed only two EGFR mutations, EGFR amplification in 17/47 samples, eight KRAS mutations, and five EML4‐ALK translocations. Although absence of rash was associated with shorter time to progression (TTP), disease‐control rate, TTP, and 1‐year survival were not different between the two dose groups, indicating the dose‐to‐rash strategy failed to increase clinical benefit. Low incidence of toxicity and low erlotinib exposure suggest standardized and maximum‐allowable dosing may be suboptimal in African Americans.

Genetic alterations of RDINK4/ARF enhancer in human cancer cells

Recent identification of an enhancer element, RDINK4/ARF (RD), in the prominent INK4/ARF locus provides a novel mechanism to simultaneously regulate the transcription of p15INK4B (p15), p14ARF, and p16INK4A (p16) tumor suppressor genes. While genetic inactivation of p15, p14ARF, and p16 in human tumors has been extensively studied, little is known about genetic alterations of RD and its impact on p15, p14ARF, and p16 in human cancer. The purpose of this study was to investigate the potential existence of genetic alterations of RD in human cancer cells. DNAs extracted from 17 different cancer cell lines and 31 primary pheochromocytoma tumors were analyzed for deletion and mutation of RD using real‐time PCR and direct DNA sequencing. We found that RD was deleted in human cancer cell lines and pheochromocytoma tumors at frequencies of 41.2% (7/17) and 13.0% (4/31), respectively. While some of these RD deletion events occurred along with deletions of the entire INK4/ARF locus, other RD deletion events were independent of genetic alterations in p15, p14ARF, and p16. Furthermore, the status of RD was poorly associated with the expression of p15, p14ARF, and p16 in tested cancer cell lines and tumors. This study demonstrates for the first time that deletion of the RD enhancer is a prevalent event in human cancer cells. Its implication in carcinogenesis remains to be further explored.