Priyanka Vijay

Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma.

UNLABELLED Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. SIGNIFICANCE We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973.

Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics

SUMMARY The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station’s history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

Distinct evolution and dynamics of epigenetic and genetic heterogeneity in acute myeloid leukemia

Genetic heterogeneity contributes to clinical outcome and progression of most tumors, but little is known about allelic diversity for epigenetic compartments, and almost no data exist for acute myeloid leukemia (AML). We examined epigenetic heterogeneity as assessed by cytosine methylation within defined genomic loci with four CpGs (epialleles), somatic mutations, and transcriptomes of AML patient samples at serial time points. We observed that epigenetic allele burden is linked to inferior outcome and varies considerably during disease progression. Epigenetic and genetic allelic burden and patterning followed different patterns and kinetics during disease progression. We observed a subset of AMLs with high epiallele and low somatic mutation burden at diagnosis, a subset with high somatic mutation and lower epiallele burdens at diagnosis, and a subset with a mixed profile, suggesting distinct modes of tumor heterogeneity. Genes linked to promoter-associated epiallele shifts during tumor progression showed increased single-cell transcriptional variance and differential expression, suggesting functional impact on gene regulation. Thus, genetic and epigenetic heterogeneity can occur with distinct kinetics likely to affect the biological and clinical features of tumors.

Modern Methods for Delineating Metagenomic Complexity

We appreciate the comments of Ackelsberg et al. (Ackelsberg et al., 2015xAckelsberg, J., Rakeman, J., Hughes, S., Peterson, J., Mead, P., Schriefer, M., Kingry, L., Hoffmaster, A., and Gee, J. Cell Syst. 2015; 1: 4–5Abstract | Full Text | Full Text PDF | Scopus (1)See all ReferencesAckelsberg et al., 2015) and have decided to revise the paper (Afshinnekoo et al., 2015xAfshinnekoo, E., Meydan, C., Chowdhury, S., Jaroudi, D., Boyer, C., Bernstein, N., Maritz, J.M., Reeves, D., Gandara, J., Chhangawala, S. et al. Cell Syst. 2015; 1: 72–87Abstract | Full Text | Full Text PDF | Scopus (23)See all ReferencesAfshinnekoo et al., 2015) as follows:Figure 3B has been corrected to show the general coverage of the Yersinia pestis pMT1 plasmid, but not the murine toxin gene (yMT). The initial claim of “…consistent 20× coverage across the murine toxin gene…” was erroneously based on looking at annotations from related plasmids and comparing different reference sequences. In actuality no reads mapped to the yMT gene.The result of low coverage to the Bacillus anthracis plasmids (pXO1 and pXO2) and no evidence of plcR SNP—an often defining feature of anthrax—is now reported in the Results section.The language in the Summary, Results, and Discussion has been revised, and speculative text about pathogenic organisms has been deleted. We now state that although all our metagenomic analysis tools identified reads with similarity to B. anthracis and Y. pestis sequences, there is minimal coverage to the backbone genome of these organisms, and there is no strong evidence to suggest these organisms are in fact present and no evidence of pathogenicity.Furthermore, in regards to the concerns of the culture methods we have posted subsequent details on the study website (http://www.pathomap.org/2015/04/13/culture-methods/) and below.A second culture experiment was performed to address the question of antibiotic resistance (Afshinnekoo et al., 2015xAfshinnekoo, E., Meydan, C., Chowdhury, S., Jaroudi, D., Boyer, C., Bernstein, N., Maritz, J.M., Reeves, D., Gandara, J., Chhangawala, S. et al. Cell Syst. 2015; 1: 72–87Abstract | Full Text | Full Text PDF | Scopus (23)See all ReferencesAfshinnekoo et al., 2015, Figure 4A). Bacteria were cultured in LB agar and then spread onto LB plates, after lawn growth, single colonies were picked and then plated onto antibiotic plates (kanamycin – 50 ug/ml, chloramphenicol – 35 ug/ml, and ampicillin – 100 ug/ml) and growth was assessed. Plates were incubated at 37°C. As a control, air samples were taken and cultured at every location. In all cases, these did not yield growth. The non-selective plate done last when replica plating also serves as a control. There was no quantitative confirmation of bacterial versus non-bacterial organisms, although there was no observable fungal growth in the samples. Further experiments are being done to dive deeper into the question of viability of microorganisms on the subway system as well as the presence of antibiotic-resistant bacteria.The field of metagenomics is relatively new but has great potential to serve an incredibly important role both in our understanding of the world around us—with key applications in the built environment—as well as the clinical realm. Nevertheless, there are still major hurdles and challenges that the field faces in order to realize this potential. We welcome and appreciate the discussion (http://microbe.net/2015/02/17/the-long-road-from-data-to-wisdom-and-from-dna-to-pathogen/) prompted by our study, and we anticipate that this large dataset will enable further experimentation, additional testing of taxonomic tools, and hopefully help in developing methodologies for metagenomic analysis.

Abstract IA08: Reprogramming human cancer cells in CDK4/6 inhibitor therapy

CDK4 and CDK6 drive cell cycle progression through early G1 and are frequently deregulated in human cancer. Selective inhibition of CDK4/6 with palbociclib (PD 0332991) has demonstrated exciting clinical efficacy in diverse human cancers. However, the mechanism that discriminates sensitivity from resistance to targeting CDK4/6 remains obscure. Mantle cell lymphoma (MCL) is an incurable non-Hodgkin9s lymphoma where deregulated CDK4 activity and cyclin D1 expression underlies unrestrained proliferation and disease progression. In a phase I clinical trial in recurrent MCL, we demonstrated that palbociclib alone produced a durable clinical response with a favorable toxicity profile. To investigate the mechanism for targeting CDK4/6, we have now shown in primary cancer cells that 1) inhibition of CDK4/6 leads to early G1 arrest that requires Rb, the CDK4/6 substrate; 2) prolonged early G1 arrest (pG1) sensitizes Rb-proficient cancer cells to killing by diverse clinically-relevant agents; and 3) pG1 sensitization stems from continuous expression of genes scheduled for early G1 only. This leads to an imbalance in genes expression, which is exacerbated in synchronous progression to S phase (pG1-S) after palbociclib withdrawal, due to incomplete restoration of cell cycle-coupled gene expression. In a hypothesis-driven phase Ib clinical trial, targeting CDK4 with palbociclib in sequential combination with the proteasome inhibitor bortezoimb was well tolerated (n=16). It exhibited a durable palbociclib dose-dependent clinical activity, including one complete remission for over 3 years with only one progression at the optimal dose combination (n=6). Longitudinal integrative whole transcriptome and whole exome sequencing of tumor cells isolated from serial lymph node biopsies demonstrated that palbociclib initially induced pG1 in MCL cells of all patients, regardless of copy number variation or mutation (ATM, p53). Cell cycle control by palbociclib is thus initially intact in MCL, but is insufficient to predict the clinical response. As predicted, only genes programmed for early G1 were expressed pG1, concurrent PI3K inactivation in primary MCL cells. However, This study represents the first investigation of genes that discriminate sensitivity from resistance in targeting CDK4/CDK6 in human cancer, through integrative longitudinal analysis of whole exome and whole transcriptome sequencing in concert with protein expression analysis and functional studies. Selective inhibition of CDK4 induces pG1 in all MCL patients, which apparently reprograms MCL for clinical response to bortezomib through PI3K inactivation and suppression of genes for redox homeostasis. Citation Format: Selina Chen-Kiang, Maurizio Di Liberto, Priyanka Vijay, David Chiron, Xiangao Huang, Scott Ely, Olivier Elemento, Christopher Mason, Lewis Cantley, John P. Leonard, Peter Martin. Reprogramming human cancer cells in CDK4/6 inhibitor therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA08.

Clinical Genomics: Challenges and Opportunities.

Next-generation sequencing (NGS) approaches are highly applicable to clinical studies. We review recent advances in sequencing technologies, as well as their benefits and tradeoffs, to provide an overview of clinical genomics from study design to computational analysis. Sequencing technologies enable genomic, transcriptomic, and epigenomic evaluations. Studies that use a combination of whole genome, exome, mRNA, and bisulfite sequencing are now feasible due to decreasing sequencing costs. Single-molecule sequencing increases read length, with the MinIONTM nanopore sequencer, which offers a uniquely portable option at a lower cost. Many of the published comparisons we review here address the challenges associated with different sequencing methods. Overall, NGS techniques, coupled with continually improving analysis algorithms, are useful for clinical studies in many realms, including cancer, chronic illness, and neurobiology. We, and others in the field, anticipate the clinical use of NGS approaches will continue to grow, especially as we shift into an era of precision medicine.

Abstract 3099: Longitudinal genomic and transcriptomic analysis of mantle cell lymphoma in a targeted combination trial of a selective CDK4/6 inhibitor

We used whole exome (WES) and whole transcriptome sequencing (WTS) to study progression of Mantle Cell Lymphoma (MCL) in the context of a clinical trial of targeted combination therapy of a CDK4 inhibitor PD0332991 (palbociclib) and a proteasome inhibitor bortezomib. Our longitudinal study design coupled with an integrative analysis approach enabled detailed molecular characterization of each subject9s disease. This includes multiple time points for three therapy Responders (Rs) and two Non-Responders (NRs); a primary goal was to understand genetic reasons for differential response. To that end, we identified copy number variants (CNVs) and single nucleotide variants (SNVs) specific to NRs that are supported by both WES and WTS data and implicate pathways relevant to mechanisms of drug action. CNVs were identified using multiple orthogonal methods that use read depth and genome-wide allele frequencies. These revealed a 29Mb chr9 hemizygous deletion unique to NRs. This region includes 15 interferon genes of the type 1 interferon signaling pathway (enrichment: p Other CNVs differentiating Rs and NRs are a 24Mb chr8 deletion found only in Rs and a 4Mb chr17 deletion found only in NRs, which includes p53 in a subject who also has a p53 mutation, resulting in 100% expression of the mutant allele. The described CNVs result in detectable shifts in expression of a large proportion of affected genes. Coupled with SNV data, where we identified 21 SNVs shared by NRs and absent in Rs, these results offer molecular clues to mechanisms of resistance in NRs, which we are currently pursuing with functional studies. Overall, we performed a thorough genomic and transcriptomic analysis of a longitudinal investigation of MCL patients during a targeted combination therapy phase I clinical trial and identified potential biomarkers of response spanning CNVs, SNVs, and differentially expressed genes. Citation Format: Priyanka Vijay, Pedro Blecua, Maurizio Di Liberto, David Chiron, Xiangao Huang, Olivier Elemento, Peter Martin, John P. Leonard, Christopher E. Mason, Selina Chen-Kiang. Longitudinal genomic and transcriptomic analysis of mantle cell lymphoma in a targeted combination trial of a selective CDK4/6 inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3099. doi:10.1158/1538-7445.AM2015-3099

Abstract 3095: Longitudinal integrative whole transcriptome and exome sequencing identifies genes that reprogram lymphoma cells for clinical response to CDK4/6 inhibition in combination therapy

CDK4 and CDK6 drive cell cycle progression through early G1 and are frequently deregulated in human cancer. The selective inhibitor palbociclib (PD 0332991) demonstrated exciting clinical efficacy in diverse human cancers, notably in metastatic breast cancer where it more than doubled the progression free survival of patients treated with letrozole. In mantle cell lymphoma (MCL), deregulated CDK4 and cyclin D1 expression underlies unrestrained proliferation and disease progression. In a phase I clinical trial in recurrent MCL, we showed that palbociclib produced a durable clinical response. However, the mechanism that discriminates sensitivity from resistance to targeting CDK4/6 remains obscure. To address this question, we demonstrated that 1) inhibition of CDK4/6 leads to early G1 arrest that requires Rb, the CDK4/6 substrate; 2) prolonged early G1 arrest (pG1) sensitizes Rb+ cancer cells to killing by diverse clinically-relevant agents; and 3) pG1 sensitization stems from continuous expression of genes scheduled for early G1 only. This sensitization is exacerbated in synchronous progression to S phase (pG1-S) after palbociclib withdrawal, due to incomplete restoration of cell cycle-coupled gene expression. In a hypothesis-driven phase Ib clinical trial we further showed that palbociclib induces pG1 and sensitizes MCL cells to the proteasome inhibitor bortezomib. At the optimal dose of palbociclib, only one in 6 patients progressed, and 4 patients had a durable responses including a complete remission for >800 days. Longitudinal integrative whole transcriptome and whole exome sequencing of tumor cells from serial lymph node biopsies demonstrated that cell cycle control by palbociclib is initially intact in Rb+ MCL but is insufficient to predict clinical response. Palbociclib induced pG1 in all patients initially regardless of the subsequent clinical response or patient-specific copy number variation or mutation (ATM, p53). As expected, pG1 maintained the expression of cell cycle genes programmed for early G1 and suppressed those scheduled for other phases, and this was reversible upon release of the early G1 block. However, expressing only genes scheduled for early G1 led to an imbalance in gene expression. Of the 1483 genes suppressed in pG1 compared with the baseline in Responders (Rs) (N = 4), 5 were up-regulated in Non-Responders (NRs) (N = 3); 11 of the 2041 genes up-regulated in pG1 in R were suppressed in NR. These oppositely expressed genes are involved in redox stress and metabolism based on functional analysis, suggesting a role for cell cycle-coupled metabolic imbalance in differential clinical response to targeting CDK4/6. Further validation of these candidate genes in the context of clinical response should advance the mechanism for therapeutic targeting of CDK4/6 as well as genome-based therapy and patient stratification. Citation Format: Maurizio Di Liberto, Peter Martin, David Chiron, Priyanka Vijay, Xiangao Huang, Pedro Blecua, Scott Ely, Olivier Elemento, John P. Leonard, Christopher E. Mason, Selina Chen-Kiang. Longitudinal integrative whole transcriptome and exome sequencing identifies genes that reprogram lymphoma cells for clinical response to CDK4/6 inhibition in combination therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3095. doi:10.1158/1538-7445.AM2015-3095