BRAF inhibition is associated with increased clonality in tumor-infiltrating lymphocytes

There have been significant advances with regard to BRAF-targeted therapies against metastatic melanoma. However, the majority of patients receiving BRAF inhibitors (BRAFi) manifest disease progression within a year. We have recently shown that melanoma patients treated with BRAFi exhibit an increase in melanoma-associated antigens and in CD8+ tumor-infiltrating lymphocytes in response to therapy. To characterize such a T-cell infiltrate, we analyzed the complementarity-determining region 3 (CDR3) of rearranged T-cell receptor (TCR) β chain-coding genes in tumor biopsies obtained before the initiation of BRAFi and 10–14 d later. We observed an increase in the clonality of tumor-infiltrating lymphocytes in 7 of 8 patients receiving BRAFi, with a statistically significant 21% aggregate increase in clonality. Over 80% of individual T-cell clones detected after initiation of BRAFi treatment were new clones. Interestingly, the comparison of tumor infiltrates with clinical responses revealed that patients who had a high proportion of pre-existing dominant clones after the administration of BRAFi responded better to therapy than patients who had a low proportion of such pre-existing dominant clones following BRAFi. These data suggest that although the inhibition of BRAF in melanoma patients results in tumor infiltration by new lymphocytes, the response to treatment appears to be related to the presence of a pre-existing population of tumor-infiltrating T-cell clones

Correspondence: Are Cognitive Functions Localizable? Colin Camerer et al. versus Marieke van Rooij and John G. Holden

The Fall 2011 issue of this journal published a two-paper section on “Neuroeconomics.” One paper, by Ernst Fehr and Antonio Rangel, clearly and concisely summarized a small part of the fast-growing literature. The second paper, “It’s about Space, It’s about Time, Neuroeconomics, and the Brain Sublime,” by Marieke van Rooij and Guy Van Orden, is beautifully written and enjoyable to read, but misleading in many critical ways. A number of economists and neuroscientists working at the intersection of the two fields shared our reaction and have signed this letter, as shown below. Some of the paper’s descriptions of empirical findings and methods in neuroeconomics are incomplete, badly out of date, or flatly wrong. In studies the authors describe in detail, their skeptical interpretations have often been refuted by published data, old and new, that they overlook

Landscape of Targeted Anti-Cancer Drug Synergies in Melanoma Identifies a Novel BRAF-VEGFR/PDGFR Combination Treatment

<div><p>A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1) transporter inhibition. We identified drugs sensitizing cell lines that are BRAF^V600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR) and platelet derived growth factor receptor (PDGFR) family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis <i>in vitro</i> and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs), demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK) kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.</p></div

BCL2A1 is a lineage-specific antiapoptotic melanoma oncogene that confers resistance to BRAF inhibition

Although targeting oncogenic mutations in the BRAF serine/threonine kinase with small molecule inhibitors can lead to significant clinical responses in melanoma, it fails to eradicate tumors in nearly all patients. Successful therapy will be aided by identification of intrinsic mechanisms that protect tumor cells from death. Here, we used a bioinformatics approach to identify drug-able, “driver” oncogenes restricted to tumor versus normal tissues. Applying this method to 88 short-term melanoma cell cultures, we show that the antiapoptotic BCL2 family member BCL2A1 is recurrently amplified in ~30% of melanomas and is necessary for melanoma growth. BCL2A1 overexpression also promotes melanomagenesis of BRAF-immortalized melanocytes. We find that high-level expression of BCL2A1 is restricted to melanoma due to direct transcriptional control by the melanoma oncogene MITF. Although BRAF inhibitors lead to cell cycle arrest and modest apoptosis, we find that apoptosis is significantly enhanced by suppression of BCL2A1 in melanomas with BCL2A1 or MITF amplification. Moreover, we find that BCL2A1 expression is associated with poorer clinical responses to BRAF pathway inhibitors in melanoma patients. Cotreatment of melanomas with BRAF inhibitors and obatoclax, an inhibitor of BCL2A1 and other BCL2 family members, overcomes intrinsic resistance to BRAF inhibitors in BCL2A1-amplified cells in vitro and in vivo. These studies identify MITF-BCL2A1 as a lineage-specific oncogenic pathway in melanoma and underscore its role for improved response to BRAF-directed therapy.National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.)National Institutes of Health (U.S.)Melanoma Research AllianceNational Institutes of Health (U.S.). (Grant eb00244

Ultra high-throughput screen to identify synergistic combinations in melanoma cells.

<p>(A) Summary of clinical development stage of 108 drugs included in the combination drug panel. (B) Example of raw UHTS data generated, demonstrating cell count information collected from DAPI channel and apoptosis data from cleaved PARP immunofluorescence; positive control of HSP90 inhibitor 17-AAG treatment is shown. (C) Summary matrix of combinatorial drug data, with each point representing the effect of one of 5,778 combinations at the standard drug concentration, as the median effect of the drug combination across all 36 melanoma cell lines on the relative cell count (left) and the calculated Bliss synergy score for that combination (right). (D) Histogram of number of cell lines a given drug combination showed synergy. Peak number of synergies were seen in one cell line, indicating many synergies are private. (E) As in (C), showing median effect of the drug combination (at standard concentration) on the relative cPARP positive proportion (left) and the calculated Bliss synergy for that cPARP level (right). (F) Graphical representation of drug combinations (drug pairs connected by an edge) that showed a significant unexpectedly high cPARP over a predicted level at the given cell count. Node size indicates the number of drug pairs that the given drug appears with other drugs on the “unexpectedly apoptotic” list. Edge color indicates the drug pair concentration (standard or low) where the elevated cPARP was found; edge pattern indicates whether the elevated cPARP was found in the setting of low cell count or normal cell count (> 80% control), with elevated cPARP in the setting of normal viability potentially representing “slow” death kinetics for that combination.</p

Cediranib synergizes with PLX4720 <i>in vivo</i>.

<p>(A) ISTMel1 xenografts were generated in immunodeficient mice (<i>n</i> = 8 per group), which were treated with PLX4720 or control chow and given cediranib or water by oral gavage. ISTMel1 xenografts showed an initial response to PLX4720 treatment alone after approximately two weeks, with some additional but non-significant response to cediranib treatment. Values are shown as mean +/- S.E.M. ranges, with significant differences in mean tumor size of both PLX4720 and PLX4720 and cediranib-treated mice compared to control treatment by ANOVA. However, the combination significantly delayed progression (defined as > 500 mm³ size) beyond this initial response (right, <i>p</i> < 0.002 between PLX4720 and PLX4720 + cediranib arms by log-rank test). (B) In contrast to ISTMel1 xenografts, RPMI7951 xenografts showed a significant difference by ANOVA test in initial response to PLX4720 versus PLX4720 and cediranib treatment after three weeks, and, right, showed a prolonged delay of progression (defined as tumor > 250 mm³) of completely PLX4720-resistant tumors (<i>p</i> < 0.0001 between PLX4720 and PLX4720 and cediranib arms by log-rank test). (C) KDR staining of tumor biopsies from patients entering clinical trials for BRAF with or without MEK inhibitors demonstrated some had strong membrane KDR staining (top) throughout the tumor (inset showing membrane staining, at arrowhead), while others were negative for KDR staining (bottom) except for expected endothelial staining. (D) Comparison of progression-free survival of patients with (<i>n</i> = 6) and without (<i>n</i> = 10) membrane KDR staining showed a significant reduction in PFS (9.3 vs. 3.8 months, <i>p</i> < 0.01 by Student’s t-test) if the patient’s biopsy expressed KDR.</p

Cytotoxic potentiation by a MDR inhibitor.

<p>(A) Screening results showing the effects at both concentrations of vincristine and lapatinib, individually, and as a combination, showing strong synergy across most melanoma cell lines as indicated by high Bliss synergy scores. (B) Confirmation of the synergistic effect of the combination of lapatinib (5μM) in A375 (<i>n</i> = 19) but not WM451Lu (<i>n</i> = 14) cells. Error bars represent s.d. of measurement replicates. (C) Representative flow cytometry data showing lapatinib potentiates G₂/M shift of A375 cell population consistent with increased vincristine effect. (D) Log₂ relative expression of the given multi-drug resistance transporter in A375 versus WM451Lu cells, showing increased MDR1 expression in A375 cells. Error bars represent s.d. of measurement replicates (<i>n</i> = 9). (E) Calcein dye flux experiments showing increased fluorescence intensity (indicating decreased MDR flux) in the presence of lapatinib or control MDR inhibitor verapamil (both at 5μM); quantitation of cell grey values shown at left. Error bars represent s.d. of measurement replicates (<i>n</i> = 4, > 200 cells per replicate). (F) MDR1 knockdown by siRNA causes a synergistic effect on cell viability in the presence of 5nM vincristine. Error bars represent s.d. of measurement replicates (<i>n</i> = 7). (G) Overexpression of MDR1 (compared to GFP control) in WM451Lu decreases sensitivity to vincristine, an effect reversible with 5μM lapatinib. Error bars represent s.d. of measurement replicates (<i>n</i> = 4)</p