Christopher Carpenter

BET inhibitor resistance emerges from leukaemia stem cells

Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL–AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.

A DNA Hypomethylation Signature Predicts Antitumor Activity of LSD1 Inhibitors in SCLC

Epigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discoveryxa0and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity.

Correlation of PD-L1 Tumor Expression and Treatment Outcomes in Patients with Renal Cell Carcinoma Receiving Sunitinib or Pazopanib: Results from COMPARZ, a Randomized Controlled Trial

Purpose: The interaction of programmed death-1 ligand (PD-L1) with its receptor (PD-1) on T cells inactivates antitumor immune responses. PD-L1 expression has been associated with poor outcomes in renal cell carcinoma (RCC) but has not been investigated in advanced RCC patients receiving VEGF-targeted therapy. Experimental Design: Formalin-fixed paraffin-embedded specimens were collected at baseline from patients in the COMPARZ trial. Tumor cell PD-L1 expression by IHC was evaluated using H-score (HS). Dual PD-L1/CD68 staining was used to differentiate PD-L1 tumor expression from tumor-associated macrophages. Intratumor CD8-positive T cells were quantified morphometrically. Associations between biomarkers and survival were investigated using the log-rank test. Results: HS data were available from 453 of 1,110 patients. Sixty-four percent of patients had negative PD-L1 expression (HS = 0). Patients with HS > 55 (n = 59, 13%) had significantly shorter overall survival (OS) than those with HS ≤ 55 in both pazopanib and sunitinib arms (median 15.1 vs. 35.6 and 15.3 vs. 27.8 months, respectively, P = 0.03). In both arms, median OS was shortest in patients with HS > 55 and intratumor CD8-positive T-cell counts > 300 (9.6 and 11.9 months with pazopanib and sunitinib, respectively). Median OS in patients with HS ≤ 55 and CD8-positive T-cell counts ≤ 300 was 36.8 and 28.0 months with pazopanib and sunitinib, respectively. Progression-free survival results were similar to OS results. Conclusions: Increased tumor cell PD-L1, or PD-L1 plus tumor CD8-positive T-cell counts, were associated with shorter survival in patients with metastatic RCC receiving VEGF-targeted agents. These findings may have implications for future design of randomized clinical trials in advanced RCC. Clin Cancer Res; 21(5); 1071–7. ©2014 AACR.

The role of aberrant VHL/HIF pathway elements in predicting clinical outcome to pazopanib therapy in patients with metastatic clear-cell renal cell carcinoma

Purpose: Inactivation of von Hippel-Lindau (VHL) gene in clear-cell renal cell carcinoma (RCC) leads to increased levels of hypoxia-inducible factors (HIF) and overexpression of HIF target genes, such as VEGF and others. VEGF-targeted agents are standard in advanced clear-cell RCC but biomarkers of activity are lacking. Experimental Design: We analyzed tumor tissue samples from metastatic clear-cell RCC patients who received pazopanib as part of clinical trial VEG102616. We evaluated several components of the VHL/HIF pathway: VHL gene inactivation (mutation and/or methylation), HIF-1α and HIF-2α immunohistochemistry staining, and HIF-1α transcriptional signature. We evaluated the association of these biomarkers with best overall response rate (ORR) and progression-free survival (PFS) to pazopanib, a standard first-line VEGF-targeted agent. Results: The VEG102616 trial enrolled 225 patients, from whom 78 samples were available for tumor DNA extraction. Of these, 70 patients had VHL mutation or methylation. VHL gene status did not correlate with ORR or PFS. Similarly, HIF-1α (65 samples) and HIF-2α (66 samples) protein levels (high vs. low) did not correlate with ORR or PFS to pazopanib. The HIF-1α transcriptional signature (46 samples) was enriched in tumors expressing high HIF-1α levels. However, the HIF-1α gene expression signature was not associated with clinical outcome to pazopanib. Conclusions: In patients with advanced clear-cell RCC, several potential biomarkers along the VHL/HIF-1α/HIF-2α axis were not found to be predictive for pazopanib activity. Additional efforts must continue to identify biomarkers associated with clinical outcome to VEGF-targeted agents in metastatic RCC. Clin Cancer Res; 19(18); 5218–26. ©2013 AACR.

Functional interdependence of BRD4 and DOT1L in MLL leukemia

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.

HLA-B*57:01 Confers Susceptibility to Pazopanib-Associated Liver Injury in Patients With Cancer

Purpose: Pazopanib is an effective treatment for advanced renal cell carcinoma and soft-tissue sarcoma. Transaminase elevations have been commonly observed in pazopanib-treated patients. We conducted pharmacogenetic analyses to explore mechanistic insight into pazopanib-induced liver injury. Experimental Design: The discovery analysis tested association between four-digit HLA alleles and alanine aminotransferase (ALT) elevation in pazopanib-treated patients with cancer from eight clinical trials (N = 1,188). We conducted confirmatory analysis using an independent dataset of pazopanib-treated patients from 23 additional trials (N = 1,002). Genome-wide association study (GWAS) for transaminase elevations was also conducted. Results: The discovery study identified an association between HLA-B*57:01 carriage and ALT elevation [P = 5.0 × 10−5 for maximum on-treatment ALT (MaxALT); P = 4.8 × 10−4 for time to ALT > 3× upper limit of normal (ULN) event; P = 4.1 × 10−5 for time to ALT > 5× ULN event] that is significant after adjustment for number of HLA alleles tested. We confirmed these associations with time to ALT elevation event (P = 8.1 × 10−4 for ALT > 3× ULN, P = 9.8 × 10−3 for ALT > 5× ULN) in an independent dataset. In the combined data, HLA-B*57:01 carriage was associated with ALT elevation (P = 4.3 × 10−5 for MaxALT, P = 5.1 × 10−6 for time to ALT > 3×ULN event, P = 5.8 × 10−6 for time to ALT > 5× ULN event). In HLA-B*57:01 carriers and noncarriers, frequency of ALT > 3× ULN was 31% and 19%, respectively, and frequency of ALT > 5× ULN was 18% and 10%, respectively. GWAS revealed a possible borderline association, which requires further evaluation. Conclusions: These data indicate that HLA-B*57:01 carriage confers higher risk of ALT elevation in patients receiving pazopanib and provide novel insight implicating an immune-mediated mechanism for pazopanib-associated hepatotoxicity in some patients. Clin Cancer Res; 22(6); 1371–7. ©2015 AACR.

Hyperbilirubinemia in pazopanib- or sunitinib-treated patients in COMPARZ is associated with UGT1A1 polymorphisms

Pazopanib and sunitinib are angiogenesis inhibitors approved for treatment of advanced renal cell carcinoma. Treatmentassociated elevations in alanine aminotransferase (ALT) and bilirubin have been reported for both therapies [1]. UGT1A1 polymorphisms are known to cause reduced expression/ activity of UGT1A1 and predispose individuals to Gilbert’s syndrome, a benign form of episodic jaundice [2, 3]. Our analyses of previous pazopanib clinical trials found that UGT1A1 *28 was associated with on-treatment hyperbilirubinemia [4]. This study extended our previous analysis of UGT1A1*28 to additional alleles (*36, *37, and *6) and investigated their association with on-therapy serum total bilirubin in COMPARZ (NCT00720941 and NCT01147822), a phase III, randomized, clinical trial comparing pazopanib versus sunitinib for the treatment of advanced renal cell carcinoma [1]. Of 1110 randomized participants, on-therapy hyperbilirubinemia [total bilirubin ≥1.5 × upper limit of the normal range (ULN)] was observed in 16% (89/557) of pazopanib-arm patients and 9% (49/553) of sunitinib-arm patients. In the pharmacogenetic population (patients who received at least one dose of study treatment, consented, and were successfully genotyped, n = 719), the incidence of hyperbilirubinemia was 17% (62 of 369) for pazopanib and 10% (34 of 350) for sunitinib. The incidence of hyperbilirubinemia varies by UGT1A1 genotypes for both therapies (Figure 1). Logistic regression adjusted for ancestry principal components was used to compare hyperbilirubinemia cases (pazopanib n = 62; sunitinib n = 34) against controls (pazopanib n = 211; sunitinib n = 212). Controls were defined as patients with all on-therapy bilirubin ≤1 × ULN and treatment exposure equal to or greater than median exposure until bilirubin elevation in cases (defined in the supplementary materials, available at Annals of Oncology online). Patients with predicted reduced UGT1A1 function (homozygous or inferred compound heterozygous for *28, *37, and *6) had higher baseline bilirubin and were more likely to experience hyperbilirubinemia when receiving either pazopanib (P = 7.7 × 10) or sunitinib (P = 1.7 × 10), with odds ratios (95% confidence interval) 9.97 (4.13–24.03) and 5.83 (2.04–16.68), respectively. After adjusting for baseline bilirubin, patients with predicted, reduced UGT1A1 function remained more likely to experience hyperbilirubinemia when receiving pazopanib (P = 0.012) or sunitinib (P = 0.024), with odds ratios (95% confidence interval) 3.65 (1.31–10.16) and 4.51 (1.26–16.11), respectively. UGT1A1 genotypes were not significantly associated with ALT ≥3 × ULN in pazopanib-treated patients. A borderline significant association (P = 0.030) was seen between UGT1A1 genotypes with reduced function (predicted) and decreased incidence of ALT ≥3 × ULN in sunitinib-treated patients; additional studies are required to confirm this observation. Pazopanib is a potent inhibitor of UGT1A1 in vitro [4]. Our data suggest that hyperbilirubinemia in pazopanibtreated patients may be the result of inhibition of UGT1A1 combined with effects of genetic variants in the UGT1A1 gene. We expect this would result in higher levels of unconjugated hyperbilirubinemia, usually associated with a benign clinical course. Sunitinib, however, does not inhibit UGT1A1 [5]. To our knowledge, this is the first study reporting the association between UGT1A1 genotype and hyperbilirubinemia in patients receiving sunitinib. Our data suggest that some instances of hyperbilirubinemia in patients treated with pazopanib or sunitinib may be benign manifestations of Gilbert’s syndrome. Bilirubin fractionation or, if not available, UGT1A1 genotyping, would enable further characterization of liver safety risk and help in making treatment decisions.

Characterisation of liver chemistry abnormalities associated with pazopanib monotherapy: a systematic review and meta-analysis of clinical trials in advanced cancer patients.

Drug-induced liver chemistry abnormalities, primarily transaminase elevations, are commonly observed in pazopanib-treated patients. This meta-analysis characterises liver chemistry abnormalities associated with pazopanib. Data of pazopanib-treated patients from nine prospective trials were integrated (N=2080). Laboratory datasets were used to characterise the incidence, timing, recovery and patterns of liver events, and subsequent rechallenge with pazopanib. Severe cases of liver chemistry abnormalities were clinically reviewed. Multivariate analyses identified predisposing factors. Twenty percent of patients developed elevated alanine aminotransferase (ALT) >3×ULN. Incidence of peak ALT >3-5×ULN, >5-8×ULN, >8-20×ULN and >20×ULN was 8%, 5%, 5% and 1%, respectively. Median time to onset for all events was 42days; 91% of events were observed within 18weeks. Recovery rates based on peak ALT >3-5×ULN, >5-8×ULN, >8-20×ULN and >20×ULN were 91%, 90%, 90% and 64%, respectively. Median time from onset to recovery was 30days, but longer in patients without dose interruption. Based on clinical review, no deaths were associated with drug-induced liver injury. Overall, 38% of rechallenged patients had ALT elevation recurrence, with 9-day median time to recurrence. Multivariate analysis showed that older age was associated with development of ALT >8×ULN. There was no correlation between hypertension and transaminitis. Our data support the current guidelines on regular liver chemistry tests after initiation of pazopanib, especially during the first 9 or 10weeks, and also demonstrate the safety of rechallenge with pazopanib.

Is change in blood pressure a biomarker of pazopanib and sunitinib efficacy in advanced/metastatic renal cell carcinoma?

AIMnPazopanib, an oral antiangiogenic agent, is associated with improved outcomes in patients with metastatic renal cell carcinoma. In this retrospective analysis, we explore hypertension, an on-target adverse event, as a predictive marker.nnnMETHODSnData from the pazopanib arm of the phase III COMPARZ trial (NCT00720941) comprised the test set. Pooled data from phase II (NCT00244764) and III (NCT00334282) pazopanib trials comprised the validation set. Data from the sunitinib arm of COMPARZ were analysed separately. Measures of efficacy were response rate, progression-free survival (PFS), and overall survival (OS). Mean arterial blood pressure (MAP) was the primary metric, and systolic hypertension (S-HTN) and diastolic hypertension (D-HTN) were secondary metrics; 4- and 12-week landmark analyses were performed.nnnRESULTSnAnalyses revealed no significant associations at the landmarks between response and MAP. We observed a trend towards improved PFS with S-HTN at week 4 (hazard ratio [HR]xa0=xa00.79, Pxa0=xa00.060) and week 12 (HRxa0=xa00.75, Pxa0=xa00.073) among pazopanib-treated patients in COMPARZ. This trend was not confirmed at week 12 in the validation set or in sunitinib-treated patients. In the test set, there was a trend towards increased OS in patients with S-HTN by week 4 (HRxa0=xa00.76, Pxa0=xa00.062)xa0and with D-HTN by week 4 (HRxa0=xa00.71, Pxa0=xa00.016) but not by week 12. No significant differences in OS were observed in sunitinib-treated patients for S-HTN or D-HTN.nnnCONCLUSIONnNeither hypertension nor any blood pressure elevation above baseline was associated with efficacy outcomes of pazopanib or sunitinib. Accordingly, management of tyrosine kinase inhibitor-induced hypertension is unlikely to compromise outcome.

Structure-Based Design of a Novel SMYD3 Inhibitor that Bridges the SAM-and MEKK2-Binding Pockets

SMYD3 is a lysine methyltransferase overexpressed in colorectal, breast, prostate, and hepatocellular tumors, and has been implicated as an oncogene in human malignancies. Methylation of MEKK2 by SMYD3 is important for regulation of the MEK/ERK pathway, suggesting the possibility of selectively targeting SMYD3 in RAS-driven cancers. Structural and kinetic characterization of SMYD3 was undertaken leading to a co-crystal structure of SMYD3 with a MEKK2-peptide substrate bound, and the observation that SMYD3 follows a partially processive mechanism. These insights allowed for the design of GSK2807, a potent and selective, SAM-competitive inhibitor of SMYD3 (Kixa0= 14xa0nM). A high-resolution crystal structure reveals that GSK2807 bridges the gap between the SAM-binding pocket and the substrate lysine tunnel of SMYD3. Taken together, our data demonstrate that small-molecule inhibitors of SMYD3 can be designed to prevent methylation of MEKK2 and these could have potential use as anticancer therapeutics.