INTEGRATIVE CANCER IMMUNOGENOMIC ANALYSIS OF SERIAL MELANOMA BIOPSIES REVEALS CORRELATES OF RESPONSE AND RESISTANCE TO SEQUENTIAL CTLA-4 AND PD-1 BLOCKADE TREATMENT

Melanoma is the most malignant form of skin cancer. The five-year survival rate for metastatic melanoma is 19.9%. Although targeted therapy of BRAF and MEK inhibitors were developed for melanoma, resistance to therapy is inevitable. Immune checkpoint blockade, which reverses the suppression of the immune system, on the other hand, has shown a durable response in 20-30% of patients with metastatic melanoma. However, more predictive and robust biomarkers of response to this therapy are still needed, and resistance mechanisms remain incompletely understood. To address this, we examined a cohort of metastatic melanoma patients treated with sequential checkpoint blockade against cytotoxic T lymphocyte antigen–4 (CTLA-4) followed by programmed death receptor–1 (PD-1) by immunogenomic profile analyses from serial tumor biopsies. From immune profiling (12 marker immunohistochemistry and NanoString Gene Expression Profiling), we found that adaptive immune signatures in tumor biopsies obtained from early on-treatment time points are predictive of response to immune checkpoint blockade. We also demonstrated differential mechanistic signatures of tumor microenvironment induced by CTLA-4 and PD-1 blockade. Importantly, VEGFA was identified as a potential target of combination therapy withPD-1blockade. From genomic profiling (whole exome sequencing and T cell receptor sequencing), we demonstrated that a higher TCR clonality in pre-treatment biopsy was predictive of response to PD-1 but notCTLA-4blockade. We also observed increased TCR clonality after CTLA-4 blockade treatment in patients responding to the following PD-1 blockade treatment. Analysis of copy number alterations (CNAs) identified a higher burden of copy number loss in nonresponders to CTLA-4 and PD-1 blockade and found that it was associated with decreased expression of genes in immune-related pathways. The effect of mutational load and burden of copy number loss on response was nonredundant, suggesting the potential utility of these as a combinatorial biomarker to optimize patient care with checkpoint blockade therapy. In summary, our integrative cancer immunogenomic analysis shows that genomic and immune profiling of longitudinal tumor biopsies can identify novel biomarkers and resistance mechanisms of immune checkpoint blockade

A preexisting rare PIK3CA e545k subpopulation confers clinical resistance to MEK plus CDK4/6 inhibition in NRAS melanoma and is dependent on S6K1 signaling

Combined MEK and CDK4/6 inhibition (MEKi + CDK4i) has shown promising clinical outcomes in patients with NRAS- mutant melanoma. Here, we interrogated longitudinal biopsies from a patient who initially responded to MEKi + CDK4i therapy but subsequently developed resistance. Whole-exome sequencing and functional validation identified an acquired PIK3CA E545K mutation as conferring drug resistance. We demonstrate that PIK3CA E545K preexisted in a rare subpopulation that was missed by both clinical and research testing, but was revealed upon multiregion sampling due to PIK3CA E545K being nonuniformly distributed. This resistant population rapidly expanded after the initiation of MEKi + CDK4i therapy and persisted in all successive samples even after immune checkpoint therapy and distant metastasis. Functional studies identified activated S6K1 as both a key marker and specific therapeutic vulnerability downstream of PIK3CA E545K -induced resistance. These results demonstrate that difficult-to-detect preexisting resistance mutations may exist more often than previously appreciated and also posit S6K1 as a common downstream therapeutic nexus for the MAPK, CDK4/6, and PI3K pathways. SIGNIFICANCE: We report the first characterization of clinical acquired resistance to MEKi + CDK4i, identifying a rare preexisting PIK3CA E545K subpopulation that expands upon therapy and exhibits drug resistance. We suggest that single-region pretreatment biopsy is insufficient to detect rare, spatially segregated drug-resistant subclones. Inhibition of S6K1 is able to resensitize PIK3CA E545K -expressing NRAS-mutant melanoma cells to MEKi + CDK4i. © 2018 AAC

The Natural Anticancer Agent Plumbagin Induces Potent Cytotoxicity in MCF-7 Human Breast Cancer Cells by Inhibiting a PI-5 Kinase for ROS Generation

<div><p>Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast <em>Schizosaccharomyces pombe</em>, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI₅₀; 10 µM) and induced cell elongation and septum formation in wild-type <em>S. pombe</em>. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in <em>S. pombe</em>. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in <em>S. pombe</em> deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.</p> </div

Comparative analysis of DIH by plumbagin between an its3-deletion mutant and a PI3K-deletion mutant.

<p>All <i>S. pombe</i> cells, including the wild-type and mutants indicated, were treated under the same conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045023#pone-0045023-g003" target="_blank">Figure 3</a> with various doses of plumbagin for 14 h, and the relative cell mass was analyzed by measuring OD₆₀₀. Data represent the mean ± standard error (n = 3).</p

ROS-dependent growth inhibition of wild-type <i>S. pombe</i> (SP286) by plumbagin.

<p>(A) Wild-type <i>S. pombe</i> cells were plated onto 96-well plates at 1×10⁵ cells/well, followed by treatment with various concentrations of plumbagin and incubation at 30°C for 14 h. Then, OD₆₀₀ was measured using a microplate reader. Data represent the mean ± standard error (n = 3). (B) <i>S. pombe</i> cells were left untreated or were treated with 10 µM plumbagin and incubated for 6 h. Then, cells were photographed. The scale bar below the right figure represents 10 µm. (C) Cells were treated with 0 or 10 µM plumbagin for 6 h and were stained with 4 µM DHE, an ROS staining reagent, for 30 min. Then, the level of ROS was measured by fluorescence microscopy as indicated in Materials and Methods. (D) Wild-type <i>S. pombe</i> cells were treated with 10 µM plumbagin in the absence or presence of NAC (2 mM) for 14 h, then the cell mass was measured as OD₆₀₀ in a microplate reader. Data represent the mean ± standard error (n = 3). ***<i>P</i><0.001 compared with the untreated samples.</p

Induction of drug-induced haploinsufficiency by plumbagin in an its3-deleted heterozygous mutant.

<p>(A) Wild-type <i>S. pombe</i> (SP286) and an its3-deleted heterozygous mutant (SPAC19G12.14; its3::KanMX) were treated with 10 µM plumbagin under the same conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045023#pone-0045023-g003" target="_blank">Figure 3</a>. Cultures were incubated at 30°C, and OD₆₀₀ was recorded every 2 h for 14 h using a microplate reader. (B) Relative cell mass at 14 h after the treatment in (A) was measured as OD₆₀₀. Data represent the mean ± standard error (n = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 compared with the untreated samples. Cells grown in the presence of 10 µM plumbagin for 14 h were either serially four-fold diluted, spotted onto YES plates, and incubated for 3 days and photographed when colonies appeared (C) or observed under microscope (D). (E) After the treatment in D, mRNA was prepared and the levels of mRNAs of its3 and Act1 were measured using RT-PCR analysis. RT-PCR analysis was performed according to the following procedures. Total RNA was isolated by lysis with glass beads in the presence of Accuzol (BIONEER). RT-PCR amplification of total RNA (1 µg/reaction) was performed using <i>its3</i> -specific primers (forward, 5′-GATGGCATTCCCCCCGATATTG-3; reverse, 5′-TCGTCGAGTTCCCTTCCTAGGG-3′) and <i>act1</i>-specific primers (forward, 5′- CACCCTTGCTTGTTGACTGAGGC-3; reverse, 5′-AGCTTCAGGGGCACGGAAACGC-3′) in a 20 -µl reaction using the Accupower RT/PCR PreMix (Bioneer, Daejeon, Korea). PCR amplification was performed as follows: one cycle at 42°C for 1 h, 94°C for 5 min, 30 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, and one final extension cycle at 72°C for 5 min. PCR reaction products were analyzed by agarose gel electrophoresis.</p

Downregulation of PI5K-1B by plumbagin and functional role of PI5K-1B for cell viability and ROS generation in MCF-7 human breast cancer cells.

<p>(A) MCF-7 cells were cultured in a 6-well plate as described in Materials and Methods and then either left untreated or treated with 5 µM plumbagin for 24 h. Then, cells were harvested, and the levels of proteins in the cell lysates were analyzed by western blotting (panel A). (B) PI5K-1B knockdown in MCF-7 cancer cells was carried out a described in Materials and Methods using PI5K-1B-specific siRNA (5′-GUCCUCAAUUAGCCAGGAA(dTdT)-3′) or a control siRNA (5′-CUUACGCUGAGUACUUCGA(dTdT)-3′) for 48 h. Then, knockdown of PI5K-1B was validated by either RT-PCR (panel Ba) or western blotting (panel Bb) as described in Materials and Methods. (C) Cell viability after siRNA transfection for 48 h was measured by the WST-1 assay. Data represent the mean ± standard error (n = 3) (panel Ca). ***<i>P</i><0.001 compared with the untreated samples. The cells after siRNA transfection for 48 h were stained with 4 µM DHE for 30 min, and then the ROS level was observed using a fluorescence microscope (excitation 518 nm, emission 605 nm) (panel Cb).</p

Diagram depicting mode of action of plumbagin.

<p>Diagram depicting mode of action of plumbagin.</p

Cytotoxic effects of plumbagin on MCF-7 human breast cancer cells in an ROS-dependent manner.

<p>(A) Chemical structure of plumbagin. (B) Cells were plated onto 6-well plates at 2×10⁴ cells/well of 96-well plate. The following day, cells were treated with 10 µM of plumbagin, and incubated for 24 h. Then, Cells were then stained using the SRB and photographed (Ba). MCF-7 cells were plated onto 6-well plates at 2×10⁴ cells/well. The following day, cells were treated with 10 µM, and incubated for 5 days. Then, colony formations were observed (Bb). (C) The viability of the treated cells in cells were treated with 10 µM of plumbagin in the presence or absence of 2 mM NAC, a ROS scavenger, and incubated for 24 h. Then, cells were photographed. (D) The viability of the treated cells in (B) was measured with the WST-1 assay. Data represent the mean ± standard error (n = 3). ***<i>P</i><0.001 compared with the untreated samples.</p

<i>S. pombe</i> heterozygous deletion mutants for drug-induced haploinsufficiency.

<p><i>S. pombe</i> heterozygous deletion mutants for drug-induced haploinsufficiency.</p