Genetic subdivisions of follicular lymphoma defined by distinct coding and noncoding mutation patterns.

Follicular lymphoma (FL) accounts for ∼20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been described comprehensively. In this study, we analyzed whole-genome sequencing data from 423 patients to compare the protein coding and noncoding mutation landscapes of untransformed FL, transformed FL, and de novo DLBCL. This revealed 2 genetically distinct subgroups of FL, which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, and biological and clinical characteristics. We implemented a machine learning-derived classification approach to stratify patients with FL into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution, and we highlight the potential for this classification to predict HT from genetic features present at diagnosis.

Emerging roles for heterogeneous ribonuclear proteins in normal and malignant B cells.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are among the most abundantly expressed RNA binding proteins in the cell and play major roles in all facets of RNA metabolism. hnRNPs are increasingly appreciated as essential for mammalian B cell development by regulating the carefully ordered expression of specific genes. Due to this tight regulation of the hnRNP-RNA network, it is no surprise that a growing number of genes encoding hnRNPs have been causally associated with the onset or progression of many cancers, including B cell neoplasms. Here we discuss our current understanding of hnRNP-driven regulation in normal, perturbed, and malignant B cells, and the most recent and emerging therapeutic innovations aimed at targeting the hnRNP-RNA network in lymphoma.

Evolutionary conservation of systemic and reversible amyloid aggregation.

In response to environmental stress, human cells have been shown to form reversible amyloid aggregates within the nucleus, termed amyloid bodies (A-bodies). These protective physiological structures share many of the biophysical characteristics associated with the pathological amyloids found in Alzheimer's and Parkinson's disease. Here, we show that A-bodies are evolutionarily conserved across the eukaryotic domain, with their detection in Drosophila melanogaster and Saccharomyces cerevisiae marking the first examples of these functional amyloids being induced outside of a cultured cell setting. The conditions triggering amyloidogenesis varied significantly among the species tested, with results indicating that A-body formation is a severe, but sublethal, stress response pathway that is tailored to the environmental norms of an organism. RNA-sequencing analyses demonstrate that the regulatory low-complexity long non-coding RNAs that drive A-body aggregation are both conserved and essential in human, mouse and chicken cells. Thus, the identification of these natural and reversible functional amyloids in a variety of evolutionarily diverse species highlights the physiological significance of this protein conformation, and will be informative in advancing our understanding of both functional and pathological amyloid aggregation events. This article has an associated First Person interview with the first author of the paper.

Coding and noncoding drivers of mantle cell lymphoma identified through exome and genome sequencing.

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.

Epigenetic Silencing of TAP1 in Aldefluor+ Breast Cancer Stem Cells Contributes to Their Enhanced Immune Evasion.

Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor+ population was expanded, suggesting inherent resistance mechanisms. Gene expression analysis of the sorted tumor cells revealed that the Aldefluor+ tumor cells has decreased expression of transporter associated with antigen processing (TAP) genes and co-stimulatory molecule CD80, which would decrease susceptibility to T cells. Similarly, the Aldefluor+ population of patient tumors and 4T1 murine mammary cells had decreased expression of TAP and co-stimulatory molecule genes. In contrast, breast CSCs identified by CD44+ CD24- do not have decreased expression of these genes, but do have increased expression of C-X-C chemokine receptor type 4. Decitabine treatment and bisulfite pyrosequencing suggests that DNA hypermethylation contributes to decreased TAP gene expression in Aldefluor+ CSCs. TAP1 knockdown resulted in increased tumor growth of 4T1 cells in immunocompetent mice. Together, this suggests immune evasion mechanisms in breast CSCs are marker specific and epigenetic silencing of TAP1 in Aldefluor+ breast CSCs contributes to their enhanced survival under immune pressure. Stem Cells 2018;36:641-654.

Hide-and-seek: the interplay between cancer stem cells and the immune system.

The enhanced ability of cancer stem cells (CSCs) to give rise to new tumors suggests that these cells may also have an advantage in evading immune detection and elimination. This tumor-forming ability, combined with the known plasticity of the immune system, which can play both protumorigenic and antitumorigenic roles, has motivated investigations into the interaction between CSCs and the immune system. Herein, we review the interplay between host immunity and CSCs by examining the immune-related mechanisms that favor CSCs and the CSC-mediated expansion of protumorigenic immune cells. Furthermore, we discuss immune cells, such as natural killer cells, that preferentially target CSCs and the strategies used by CSCs to evade immune detection and destruction. An increased understanding of these interactions and the pathways that regulate them may allow us to harness immune system components to create new adjuvant therapies that eradicate CSCs and improve patient survival.

Newly recruited CD11b+, GR-1+, Ly6C(high) myeloid cells augment tumor-associated immunosuppression immediately following the therapeutic administration of oncolytic reovirus.

Tumor-associated immunosuppression aids cancer cells to escape immune-mediated attack and subsequent elimination. Recently, however, many oncolytic viruses, including reovirus, have been reported to overturn such immunosuppression and promote the development of a clinically desired antitumor immunity, which is known to promote favorable patient outcomes. Contrary to this existing paradigm, in this article we demonstrate that reovirus augments tumor-associated immunosuppression immediately following its therapeutic administration. Our data show that reovirus induces preferential differentiation of highly suppressive CD11b(+), Gr-1(+), Ly6C(high) myeloid cells from bone marrow hematopoietic progenitor cells. Furthermore, reovirus administration in tumor-bearing hosts drives time-dependent recruitment of CD11b(+), Gr-1(+), Ly6C(high) myeloid cells in the tumor milieu, which is further supported by virus-induced increased expression of numerous immune factors involved in myeloid-derived suppressor cell survival and trafficking. Most importantly, CD11b(+), Gr-1(+), Ly6C(high) myeloid cells specifically potentiate the suppression of T cell proliferation and are associated with the absence of IFN-γ response in the tumor microenvironment early during oncotherapy. Considering that the qualitative traits of a specific antitumor immunity are largely dictated by the immunological events that precede its development, our findings are of critical importance and must be considered while devising complementary interventions aimed at promoting the optimum efficacy of oncolytic virus-based anticancer immunotherapies.

ALDH1A3 is the switch that determines the balance of ALDH+ and CD24-CD44+ cancer stem cells, EMT-MET, and glucose metabolism in breast cancer.

Plasticity is an inherent feature of cancer stem cells (CSCs) and regulates the balance of key processes required at different stages of breast cancer progression, including epithelial-to-mesenchymal transition (EMT) versus mesenchymal-to-epithelial transition (MET), and glycolysis versus oxidative phosphorylation. Understanding the key factors that regulate the switch between these processes could lead to novel therapeutic strategies that limit tumor progression. We found that aldehyde dehydrogenase 1A3 (ALDH1A3) regulates these cancer-promoting processes and the abundance of the two distinct breast CSC populations defined by high ALDH activity and CD24-CD44+ cell surface expression. While ALDH1A3 increases ALDH+ breast cancer cells, it inversely suppresses the CD24-CD44+ population by retinoic acid signaling-mediated gene expression changes. This switch in CSC populations induced by ALDH1A3 was paired with decreased migration but increased invasion and an intermediate EMT phenotype. We also demonstrate that ALDH1A3 increases oxidative phosphorylation and decreases glycolysis and reactive oxygen species (ROS). The effects of ALDH1A3 reduction were countered with the glycolysis inhibitor 2-deoxy-D-glucose (2DG). In cell culture and tumor xenograft models, 2DG suppresses the increase in the CD24-CD44+ population and ROS induced by ALDH1A3 knockdown. Combined inhibition of ALDH1A3 and glycolysis best reduces breast tumor growth and tumor-initiating cells, suggesting that the combination of targeting ALDH1A3 and glycolysis has therapeutic potential for limiting CSCs and tumor progression. Together, these findings identify ALDH1A3 as a key regulator of processes required for breast cancer progression and depletion of ALDH1A3 makes breast cancer cells more susceptible to glycolysis inhibition.

STAT6 mutations enriched at diffuse large B-cell lymphoma relapse reshape the tumor microenvironment.

Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin's lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6- tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.

GLA-SE, a synthetic toll-like receptor 4 agonist, enhances T-cell responses to influenza vaccine in older adults.

BACKGROUND: The decline in influenza vaccine efficacy in older adults is associated with a limited ability of current split-virus vaccines (SVVs) to stimulate cytotoxic T lymphocyte (CTL) responses required for clinical protection against influenza. METHODS: The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) was combined with SVV to stimulate peripheral blood mononuclear cells (PBMCs) in vitro to determine the cytokine response in dendritic cell subsets. Stimulated PBMCs were then challenged with live influenza virus to mimic the response to natural infection following vaccination, using previously identified T-cell correlates of protection. RESULTS: GLA-SE significantly increased the proportion of myeloid dendritic cells that produced tumor necrosis factor α, interleukin 6, and interleukin 12. When combined with SVV to stimulate PBMCs in vitro, this effect of GLA-SE was shown to regulate a T-helper 1 cell response upon challenge with live influenza virus; interleukin 10 production was suppressed, thus significantly increasing the interferon γ to interleukin 10 ratio and the cytolytic (granzyme B) response to influenza virus challenge, both of which have been shown to correlate with protection against influenza in older adults. CONCLUSIONS: Our findings suggest that a novel adjuvant, GLA-SE, combined with standard SVV has the potential to significantly improve vaccine-mediated protection against influenza in older adults.

Exploiting the fibroblast growth factor receptor-1 vulnerability to therapeutically restrict the MYC-EZH2-CDKN1C axis-driven proliferation in Mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a lethal hematological malignancy with a median survival of 4 years. Its lethality is mainly attributed to a limited understanding of clinical tumor progression and resistance to current therapeutic regimes. Intrinsic, prolonged drug treatment and tumor-microenvironment (TME) facilitated factors impart pro-tumorigenic and drug-insensitivity properties to MCL cells. Hence, elucidating neoteric pharmacotherapeutic molecular targets involved in MCL progression utilizing a global "unified" analysis for improved disease prevention is an earnest need. Using integrated transcriptomic analyses in MCL patients, we identified a Fibroblast Growth Factor Receptor-1 (FGFR1), and analyses of MCL patient samples showed that high FGFR1 expression was associated with shorter overall survival in MCL patient cohorts. Functional studies using pharmacological intervention and loss of function identify a novel MYC-EZH2-CDKN1C axis-driven proliferation in MCL. Further, pharmacological targeting with erdafitinib, a selective small molecule targeting FGFRs, induced cell-cycle arrest and cell death in-vitro, inhibited tumor progression, and improved overall survival in-vivo. We performed extensive pre-clinical assessments in multiple in-vivo model systems to confirm the therapeutic potential of erdafitinib in MCL and demonstrated FGFR1 as a viable therapeutic target in MCL.

A novel cell-based screen identifies chemical entities that reverse the immune-escape phenotype of metastatic tumours.

Genetic and epigenetic events have been implicated in the downregulation of the cellular antigen processing and presentation machinery (APM), which in turn, has been associated with cancer evasion of the immune system. When these essential components are lacking, cancers develop the ability to subvert host immune surveillance allowing cancer cells to become invisible to the immune system and, in turn, promote cancer metastasis. Here we describe and validate the first high-throughput cell-based screening assay to identify chemical extracts and unique chemical entities that reverse the downregulation of APM components in cell lines derived from metastatic tumours. Through the screening of a library of 480 marine invertebrate extracts followed by bioassay-guided fractionation, curcuphenol, a common sesquiterpene phenol derived from turmeric, was identified as the active compound of one of the extracts. We demonstrate that curcuphenol induces the expression of the APM components, TAP-1 and MHC-I molecules, in cell lines derived from both metastatic prostate and lung carcinomas. Turmeric and curcumins that contain curcuphenol have long been utilized not only as a spice in the preparation of food, but also in traditional medicines for treating cancers. The remarkable discovery that a common component of spices can increase the expression of APM components in metastatic tumour cells and, therefore reverse immune-escape mechanisms, provides a rationale for the development of foods and advanced nutraceuticals as therapeutic candidates for harnessing the power of the immune system to recognize and destroy metastatic cancers.

Breast Cancer Xenograft Murine Models.

Mice are used as model organisms to understand the pathological basis of a variety of human diseases, including breast cancer. Both immunocompetent and immunocompromised mouse models are used depending on the scope of the study. Immunocompetent models allow the study of the impact of the immune system in murine models of mammary cancer, while immunodeficient mice serve as ideal host organisms to understand the behavior of human breast cancers within a biological system. Xenografting of human breast cancer cells into immunocompromised mouse models continues to be the most used fundamental animal model in preclinical breast cancer research. These in vivo models allow critical understanding of tumor biology and assessment of novel treatments, a necessary prelude to testing new drugs in the clinic. In this chapter, we provide detailed methodology for the use of non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice in several breast cancer xenografting procedures, including established cell lines and patient-derived xenografts (PDXs).

Human-specific GAPDH qRT-PCR is an accurate and sensitive method of xenograft metastasis quantification.

Metastasis is the primary cause of cancer-related mortality. Experimental models that accurately reflect changes in metastatic burden are essential tools for developing treatments and to gain a better understanding of disease. Murine xenograft tumor models mimic the human scenario and provide a platform for metastasis analyses. An ex vivo quantitative method, gaining favor for its ease and accuracy, is quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR); however, it is currently unclear how well this method correlates with gold-standard histological analysis, and its use has required detection of overexpressed exogenous genes. We have introduced a variation of the qRT-PCR method: human-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDH) qRT-PCR, which allows quantification of metastasis in xenograft models without the requirement of overexpressed exogenous genes. This makes the method easily amenable to many xenograft models without alteration of the cancer cells. We determined that the method is able to detect a few human cells within abundant mouse lung tissue. Further, the human-specific GAPDH qRT-PCR is more sensitive and correlates with histological analysis in terms of determining relative metastatic burden, suggesting that human-specific GAPDH qRT-PCR could be used as a primary method for quantification of disseminated human cells in murine xenograft models.

Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells.

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1-/- mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1-/- mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1-/- compared to wild-type mice. Furthermore, cultured Uch-l1-/- SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

Mast Cell and Eosinophil Activation Are Associated With COVID-19 and TLR-Mediated Viral Inflammation: Implications for an Anti-Siglec-8 Antibody.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection represents a global health crisis. Immune cell activation via pattern recognition receptors has been implicated as a driver of the hyperinflammatory response seen in COVID-19. However, our understanding of the specific immune responses to SARS-CoV-2 remains limited. Mast cells (MCs) and eosinophils are innate immune cells that play pathogenic roles in many inflammatory responses. Here we report MC-derived proteases and eosinophil-associated mediators are elevated in COVID-19 patient sera and lung tissues. Stimulation of viral-sensing toll-like receptors in vitro and administration of synthetic viral RNA in vivo induced features of hyperinflammation, including cytokine elevation, immune cell airway infiltration, and MC-protease production-effects suppressed by an anti-Siglec-8 monoclonal antibody which selectively inhibits MCs and depletes eosinophils. Similarly, anti-Siglec-8 treatment reduced disease severity and airway inflammation in a respiratory viral infection model. These results suggest that MC and eosinophil activation are associated with COVID-19 inflammation and anti-Siglec-8 antibodies are a potential therapeutic approach for attenuating excessive inflammation during viral infections.

An in vivo genome-wide shRNA screen identifies BCL6 as a targetable biomarker of paclitaxel resistance in breast cancer.

Paclitaxel is a common breast cancer drug; however, some tumors are resistant. The identification of biomarkers for paclitaxel resistance or sensitivity would enable the development of strategies to improve treatment efficacy. A genome-wide in vivo shRNA screen was performed on paclitaxel-treated mice with MDA-MB-231 tumors to identify genes associated with paclitaxel sensitivity or resistance. Gene expression of the top screen hits was associated with tumor response (resistance or sensitivity) among patients who received neoadjuvant chemotherapy containing paclitaxel. We focused our validation on screen hit B-cell lymphoma 6 (BCL6), which is a therapeutic target in cancer but for which no effects on drug response have been reported. Knockdown of BCL6 resulted in increased tumor regression in mice treated with paclitaxel. Similarly, inhibiting BCL6 using a small molecule inhibitor enhanced paclitaxel treatment efficacy both in vitro and in vivo in breast cancer models. Mechanism studies revealed that reduced BCL6 enhances the efficacy of paclitaxel by inducing sustained G1/S arrest, concurrent with increased apoptosis and expression of target gene cyclin-dependent kinase inhibitor 1A. In summary, the genome-wide shRNA knockdown screen has identified BCL6 as a potential targetable resistance biomarker of paclitaxel response in breast cancer.

ALDH1A3-regulated long non-coding RNA NRAD1 is a potential novel target for triple-negative breast tumors and cancer stem cells.

To discover novel therapeutic targets for triple-negative breast cancer (TNBC) and cancer stem cells (CSCs), we screened long non-coding RNAs (lncRNAs) most enriched in TNBCs for high expression in CSCs defined by high Aldefluor activity and associated with worse patient outcomes. This led to the identification of non-coding RNA in the aldehyde dehydrogenase 1 A pathway (NRAD1), also known as LINC00284. Targeting NRAD1 in TNBC tumors using antisense oligonucleotides reduced cell survival, tumor growth, and the number of cells with CSC characteristics. Expression of NRAD1 is regulated by an enzyme that causes Aldefluor activity in CSCs, aldehyde dehydrogenase 1A3 (ALDH1A3) and its product retinoic acid. Cellular fractionation revealed that NRAD1 is primarily nuclear localized, which suggested a potential function in gene regulation. This was confirmed by transcriptome profiling and chromatin isolation by RNA purification, followed by sequencing (ChIRP-seq), which demonstrated that NRAD1 has enriched chromatin interactions among the genes it regulates. Gene Ontology enrichment analysis revealed that NRAD1 regulates expression of genes involved in differentiation and catabolic processes. NRAD1 also contributes to gene expression changes induced by ALDH1A3; thereby, the induction of NRAD1 is a novel mechanism through which ALDH1A3 regulates gene expression. Together, these data identify lncRNA NRAD1 as a downstream effector of ALDH1A3, and a target for TNBCs and CSCs, with functions in cell survival and regulation of gene expression.

Decitabine Response in Breast Cancer Requires Efficient Drug Processing and Is Not Limited by Multidrug Resistance.

Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene-specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

Genetic and evolutionary patterns of treatment resistance in relapsed B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) patients are typically treated with immunochemotherapy containing rituximab (rituximab, cyclophosphamide, hydroxydaunorubicin-vincristine (Oncovin), and prednisone [R-CHOP]); however, prognosis is extremely poor if R-CHOP fails. To identify genetic mechanisms contributing to primary or acquired R-CHOP resistance, we performed target-panel sequencing of 135 relapsed/refractory DLBCLs (rrDLBCLs), primarily comprising circulating tumor DNA from patients on clinical trials. Comparison with a metacohort of 1670 diagnostic DLBCLs identified 6 genes significantly enriched for mutations upon relapse. TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations remained clonally persistent throughout treatment in paired diagnostic-relapse samples, suggesting a role in primary treatment resistance. Nonsense and missense mutations affecting MS4A1, which encodes CD20, are exceedingly rare in diagnostic samples but show recurrent patterns of clonal expansion following rituximab-based therapy. MS4A1 missense mutations within the transmembrane domains lead to loss of CD20 in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. In a time series from a patient treated with multiple rounds of therapy, tumor heterogeneity and minor MS4A1-harboring subclones contributed to rapid disease recurrence, with MS4A1 mutations as founding events for these subclones. TP53 and KMT2D mutation status, in combination with other prognostic factors, may be used to identify high-risk patients prior to R-CHOP for posttreatment monitoring. Using liquid biopsies, we show the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens.