CancerCellTracker: a brightfield time-lapse microscopy framework for cancer drug sensitivity estimation.

MOTIVATION: Time-lapse microscopy is a powerful technique that relies on images of live cells cultured ex vivo that are captured at regular intervals of time to describe and quantify their behavior under certain experimental conditions. This imaging method has great potential in advancing the field of precision oncology by quantifying the response of cancer cells to various therapies and identifying the most efficacious treatment for a given patient. Digital image processing algorithms developed so far require high-resolution images involving very few cells originating from homogeneous cell line populations. We propose a novel framework that tracks cancer cells to capture their behavior and quantify cell viability to inform clinical decisions in a high-throughput manner. RESULTS: The brightfield microscopy images a large number of patient-derived cells in an ex vivo reconstruction of the tumor microenvironment treated with 31 drugs for up to 6 days. We developed a robust and user-friendly pipeline CancerCellTracker that detects cells in co-culture, tracks these cells across time and identifies cell death events using changes in cell attributes. We validated our computational pipeline by comparing the timing of cell death estimates by CancerCellTracker from brightfield images and a fluorescent channel featuring ethidium homodimer. We benchmarked our results using a state-of-the-art algorithm implemented in ImageJ and previously published in the literature. We highlighted CancerCellTracker's efficiency in estimating the percentage of live cells in the presence of bone marrow stromal cells. AVAILABILITY AND IMPLEMENTATION: https://github.com/compbiolabucf/CancerCellTracker. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Metabolic Changes Are Associated with Melphalan Resistance in Multiple Myeloma.

Multiple myeloma is an incurable hematological malignancy that impacts tens of thousands of people every year in the United States. Treatment for eligible patients involves induction, consolidation with stem cell rescue, and maintenance. High-dose therapy with a DNA alkylating agent, melphalan, remains the primary drug for consolidation therapy in conjunction with autologous stem-cell transplantation; as such, melphalan resistance remains a relevant clinical challenge. Here, we describe a proteometabolomic approach to examine mechanisms of acquired melphalan resistance in two cell line models. Drug metabolism, steady-state metabolomics, activity-based protein profiling (ABPP, data available at PRIDE: PXD019725), acute-treatment metabolomics, and western blot analyses have allowed us to further elucidate metabolic processes associated with melphalan resistance. Proteometabolomic data indicate that drug-resistant cells have higher levels of pentose phosphate pathway metabolites. Purine, pyrimidine, and glutathione metabolisms were commonly altered, and cell-line-specific changes in metabolite levels were observed, which could be linked to the differences in steady-state metabolism of naïve cells. Inhibition of selected enzymes in purine synthesis and pentose phosphate pathways was evaluated to determine their potential to improve melphalan's efficacy. The clinical relevance of these proteometabolomic leads was confirmed by comparison of tumor cell transcriptomes from newly diagnosed MM patients and patients with relapsed disease after treatment with high-dose melphalan and autologous stem-cell transplantation. The observation of common and cell-line-specific changes in metabolite levels suggests that omic approaches will be needed to fully examine melphalan resistance in patient specimens and define personalized strategies to optimize the use of high-dose melphalan.

Biodistribution and Multicompartment Pharmacokinetic Analysis of a Targeted α Particle Therapy.

Targeted α particle therapy (TAT) is ideal for treating disease while minimizing damage to surrounding nontargeted tissues due to short path length and high linear energy transfer (LET). We developed a TAT for metastatic uveal melanoma, targeting the melanocortin-1 receptor (MC1R), which is expressed in 94% of uveal melanomas. Two versions of the therapy are being investigated: 225Ac-DOTA-Ahx-MC1RL (225Ac-Ahx) and 225Ac-DOTA-di-d-Glu-MC1RL (225Ac-di-d-Glu). The biodistribution (BD) from each was studied and a multicompartment pharmacokinetic (PK) model was developed to describe drug distribution rates. Two groups of 16 severe combined immunodeficient (SCID) mice bearing high MC1R expressing tumors were intravenously injected with 225Ac-Ahx or 225Ac-di-d-Glu. After injection, four groups (n = 4) were euthanized at 24, 96, 144, and 288 h time points for each cohort. Tumors and 13 other organs were harvested at each time point. Isomeric γ spectra were measured in tissue samples using a scintillation γ detector and converted to α activity using factors for γ ray abundance per α decay. Time activity curves were calculated for each organ. A five-compartment PK model was built with the following compartments: blood, tumor, normal tissue, kidney, and liver. This model is characterized by a system of five ordinary differential equations using mass action kinetics, which describe uptake, intercompartmental transitions, and clearance rates. The ordinary differential equations were simultaneously solved and fit to experimental data using a genetic algorithm for optimization. The BD data show that both compounds have minimal distribution to organs at risk other than the kidney and liver. The PK parameter estimates had less than 5% error. From these data, 225Ac-Ahx showed larger and faster uptake in the liver. Both compounds had comparable uptake and clearance rates for other compartments. The BD and PK behavior for two targeted radiopharmaceuticals were investigated. The PK model fit the experimental data and provided insight into the kinetics of the compounds systematically.

High School Internship Program in Integrated Mathematical Oncology (HIP IMO): Five-Year Experience at Moffitt Cancer Center.

Modern cancer research, and the wealth of data across multiple spatial and temporal scales, has created the need for researchers that are well versed in the life sciences (cancer biology, developmental biology, immunology), medical sciences (oncology) and natural sciences (mathematics, physics, engineering, computer sciences). College undergraduate education traditionally occurs in disciplinary silos, which creates a steep learning curve at the graduate and postdoctoral levels that increasingly bridge multiple disciplines. Numerous colleges have begun to embrace interdisciplinary curricula, but students who double major in mathematics (or other quantitative sciences) and biology (or medicine) remain scarce. We identified the need to educate junior and senior high school students about integrating mathematical and biological skills, through the lens of mathematical oncology, to better prepare students for future careers at the interdisciplinary interface. The High school Internship Program in Integrated Mathematical Oncology (HIP IMO) at Moffitt Cancer Center has so far trained 59 students between 2015 and 2019. We report here on the program structure, training deliverables, curriculum and outcomes. We hope to promote interdisciplinary educational activities early in a student's career.

The bone ecosystem facilitates multiple myeloma relapse and the evolution of heterogeneous drug resistant disease.

Multiple myeloma (MM) is an osteolytic malignancy that is incurable due to the emergence of treatment resistant disease. Defining how, when and where myeloma cell intrinsic and extrinsic bone microenvironmental mechanisms cause relapse is challenging with current biological approaches. Here, we report a biology-driven spatiotemporal hybrid agent-based model of the MM-bone microenvironment. Results indicate MM intrinsic mechanisms drive the evolution of treatment resistant disease but that the protective effects of bone microenvironment mediated drug resistance (EMDR) significantly enhances the probability and heterogeneity of resistant clones arising under treatment. Further, the model predicts that targeting of EMDR deepens therapy response by eliminating sensitive clones proximal to stroma and bone, a finding supported by in vivo studies. Altogether, our model allows for the study of MM clonal evolution over time in the bone microenvironment and will be beneficial for optimizing treatment efficacy so as to significantly delay disease relapse.

Genomic Classification and Individualized Prognosis in Multiple Myeloma.

PURPOSE: Outcomes for patients with newly diagnosed multiple myeloma (NDMM) are heterogenous, with overall survival (OS) ranging from months to over 10 years. METHODS: To decipher and predict the molecular and clinical heterogeneity of NDMM, we assembled a series of 1,933 patients with available clinical, genomic, and therapeutic data. RESULTS: Leveraging a comprehensive catalog of genomic drivers, we identified 12 groups, expanding on previous gene expression-based molecular classifications. To build a model predicting individualized risk in NDMM (IRMMa), we integrated clinical, genomic, and treatment variables. To correct for time-dependent variables, including high-dose melphalan followed by autologous stem-cell transplantation (HDM-ASCT), and maintenance therapy, a multi-state model was designed. The IRMMa model accuracy was significantly higher than all comparator prognostic models, with a c-index for OS of 0.726, compared with International Staging System (ISS; 0.61), revised-ISS (0.572), and R2-ISS (0.625). Integral to model accuracy was 20 genomic features, including 1q21 gain/amp, del 1p, TP53 loss, NSD2 translocations, APOBEC mutational signatures, and copy-number signatures (reflecting the complex structural variant chromothripsis). IRMMa accuracy and superiority compared with other prognostic models were validated on 256 patients enrolled in the GMMG-HD6 (ClinicalTrials.gov identifier: NCT02495922) clinical trial. Individualized patient risks were significantly affected across the 12 genomic groups by different treatment strategies (ie, treatment variance), which was used to identify patients for whom HDM-ASCT is particularly effective versus patients for whom the impact is limited. CONCLUSION: Integrating clinical, demographic, genomic, and therapeutic data, to our knowledge, we have developed the first individualized risk-prediction model enabling personally tailored therapeutic decisions for patients with NDMM.

In vivo and in silico pharmacokinetics and biodistribution of a melanocortin receptor 1 targeted agent in preclinical models of melanoma.

The melanocortin 1 receptor (MC1R) is overexpressed in most melanoma metastases, making it a promising target for imaging of melanomas. In this study, the expression of MC1R in a large fraction of patients with melanoma was confirmed using mRNA and tissue microarray. Here, we have characterized the in vivo tumor and tissue distribution and pharmacokinetics (PK) of uptake and clearance of a MC1R specific peptidomimetic ligand conjugated to a near-infrared fluorescent dye. We propose an interdisciplinary framework to bridge the different time and space scales of ligand-tumor-host interactions: intravital fluorescence microscopy to quantify probe internalization at the cellular level, a xenograft tumor model for whole body pharmacokinetics, and a computational pharmacokinetic model for integration and interpretation of experimental data. Administration of the probe into mice bearing tumors with high and low MC1R expression demonstrated normalized image intensities that correlated with expression levels (p < 0.05). The biodistribution study showed high kidney uptake as early as 30 min postinjection. The PK computational model predicted the presence of receptors in the kidneys with a lower affinity, but at higher numbers than in the tumors. As the mouse kidney is known to express the MC5R, this hypothesis was confirmed by both coinjection of a ligand with higher MC5R affinity compared to MC1R and by injection of lower probe concentrations (e.g., 1 nmol/kg), both leading to decreased kidney accumulation of the MC1R ligand. In addition, through this interdisciplinary approach we could predict the rates of ligand accumulation and clearance into and from organs and tumors, and the amount of injected ligand required to have maximum specific retention in tumors. These predictions have potential to aid in the translation of a targeted agent from lab to the clinic. In conclusion, the characterized MC1R-specific probe has excellent potential for in vivo detection of melanoma metastases. The process of cell-surface marker validation, targeted imaging probe development, and in vitro, in vivo, and in silico characterization described in this study can be generally applied to preclinical development of targeted agents.

Whole-Exome sequencing analysis identified TMSB10/TRABD2A locus to be associated with carfilzomib-related cardiotoxicity among patients with multiple myeloma.

Background: Proteasome inhibitor Carfilzomib (CFZ) is effective in treating patients with refractory or relapsed multiple myeloma (MM) but has been associated with cardiovascular adverse events (CVAE) such as hypertension, cardiomyopathy, and heart failure. This study aimed to investigate the contribution of germline genetic variants in protein-coding genes in CFZ-CVAE among MM patients using whole-exome sequencing (WES) analysis. Methods: Exome-wide single-variant association analysis, gene-based analysis, and rare variant analyses were performed on 603,920 variants in 247 patients with MM who have been treated with CFZ and enrolled in the Oncology Research Information Exchange Network (ORIEN) at the Moffitt Cancer Center. Separate analyses were performed in European Americans and African Americans followed by a trans-ethnic meta-analysis. Results: The most significant variant in the exome-wide single variant analysis was a missense variant rs7148 in the thymosin beta-10/TraB Domain Containing 2A (TMSB10/TRABD2A) locus. The effect allele of rs7148 was associated with a higher risk of CVAE [odds ratio (OR) = 9.3 with a 95% confidence interval of 3.9-22.3, p = 5.42*10-7]. MM patients with rs7148 AG or AA genotype had a higher risk of CVAE (50%) than those with GG genotype (10%). rs7148 is an expression quantitative trait locus (eQTL) for TRABD2A and TMSB10. The gene-based analysis also showed TRABD2A as the most significant gene associated with CFZ-CVAE (p = 1.06*10-6). Conclusions: We identified a missense SNP rs7148 in the TMSB10/TRABD2A as associated with CFZ-CVAE in MM patients. More investigation is needed to understand the underlying mechanisms of these associations.

CK1δ and CK1ε Signaling Sustains Mitochondrial Metabolism and Cell Survival in Multiple Myeloma.

Multiple myeloma remains an incurable malignancy due to acquisition of intrinsic programs that drive therapy resistance. Here we report that casein kinase-1δ (CK1δ) and CK1ε are therapeutic targets in multiple myeloma that are necessary to sustain mitochondrial metabolism. Specifically, the dual CK1δ/CK1ε inhibitor SR-3029 had potent in vivo and ex vivo anti-multiple myeloma activity, including against primary multiple myeloma patient specimens. RNA sequencing (RNA-seq) and metabolic analyses revealed inhibiting CK1δ/CK1ε disables multiple myeloma metabolism by suppressing genes involved in oxidative phosphorylation (OxPhos), reducing citric acid cycle intermediates, and suppressing complexes I and IV of the electron transport chain. Finally, sensitivity of multiple myeloma patient specimens to SR-3029 correlated with elevated expression of mitochondrial genes, and RNA-seq from 687 multiple myeloma patient samples revealed that increased CSNK1D, CSNK1E, and OxPhos genes correlate with disease progression and inferior outcomes. Thus, increases in mitochondrial metabolism are a hallmark of multiple myeloma progression that can be disabled by targeting CK1δ/CK1ε. SIGNIFICANCE: CK1δ and CK1ε are attractive therapeutic targets in multiple myeloma whose expression increases with disease progression and connote poor outcomes, and that are necessary to sustain expression of genes directing OxPhos.

Racial and ethnic differences in clonal hematopoiesis, tumor markers, and outcomes of patients with multiple myeloma.

Multiple myeloma (MM) incidence, mortality, and survival vary by race and ethnicity, but the causes of differences remain unclear. We investigated demographic, clinical, and molecular features of diverse MM patients to elucidate mechanisms driving clinical disparities. This study included 495 MM patients (self-reported Hispanic, n = 45; non-Hispanic Black, n = 52; non-Hispanic White, n = 398). Hispanic and non-Hispanic Black individuals had an earlier age of onset than non-Hispanic White individuals (53 and 57 vs 63 years, respectively, P < .001). There were no differences in treatment by race and ethnicity groups, but non-Hispanic Black patients had a longer time to hematopoietic cell transplant than non-Hispanic White patients (376 days vs 248 days; P = .01). Overall survival (OS) was improved for non-Hispanic Black compared with non-Hispanic White patients (HR, 0.50; 95% CI, 0.31-0.81; P = .005), although this association was attenuated after adjusting for clinical features (HR, 0.62; 95% CI, 0.37-1.03; P = .06). Tumor mutations in IRF4 were most common in Hispanic patients, and mutations in SP140, AUTS2, and SETD2 were most common in non-Hispanic Black patients. Differences in tumor expression of BCL7A, SPEF2, and ANKRD26 by race and ethnicity were observed. Clonal hematopoiesis was detected in 12% of patients and associated with inferior OS in non-Hispanic Black patients compared with patients without clonal hematopoiesis (HR, 4.36; 95% CI, 1.36-14.00). This study provides insight into differences in molecular features that may drive clinical disparities in MM patients receiving comparable treatment, with the novel inclusion of Hispanic individuals.

Replicative Instability Drives Cancer Progression.

In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress exhibit genomic instability and poor survival rates across tumor types. Seminal studies have demonstrated that genomic instability develops following inactivation of BRCA1, BRCA2, or BRCA-related genes. However, it is recognized that many tumors exhibit genomic instability but lack BRCA inactivation. We sought to identify a pan-cancer mechanism that underpins genomic instability and cancer progression in BRCA-wildtype tumors. Methods: Using multi-omics data from two independent consortia, we analyzed data from dozens of tumor types to identify patient cohorts characterized by poor outcomes, genomic instability, and wildtype BRCA genes. We developed several novel metrics to identify the genetic underpinnings of genomic instability in tumors with wildtype BRCA. Associated clinical data was mined to analyze patient responses to standard of care therapies and potential differences in metastatic dissemination. Results: Systematic analysis of the DNA repair landscape revealed that defective single-strand break repair, translesion synthesis, and non-homologous end-joining effectors drive genomic instability in tumors with wildtype BRCA and BRCA-related genes. Importantly, we find that loss of these effectors promotes replication stress, therapy resistance, and increased primary carcinoma to brain metastasis. Conclusions: Our results have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by the accumulation of intra-chromosomal, gene-level gain and loss events at replication stress sensitive (RSS) genome sites. We find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution. Clinically, we find that RIN drives therapy resistance and distant metastases across multiple tumor types.

Adaptation to survival in germinal center is the initial step in onset of indolent stage of multiple myeloma.

Aberrant mutations of centrocytes in germinal centers (GC) can generate two completely different diseases: B-cell lymphomas and monoclonal gammopathy of undetermined significance (MGUS). In this article we use computational models to examine the evolutionary dynamics by which initial adaptation to survival in the GC allows naive MGUS cells to proliferate in the bone marrow and initiate the evolutionary process that will lead to aggressive multiple myeloma (MM). Our simulations show that MGUS cells may generate bone marrow tumors ranging from indolent to aggressive, depending on the original adaptation in the GC. All these tumors, however, are limited to approximately 15% of the marrow cellularity due to hypoxia-induced quiescence (this correlates with the cellularity that separates MGUS and MM, ∼10%). Resistance to hypoxia-induced quiescence and cell death was one of the two major bone marrow adaptations that allowed continued tumor growth and establishment of paracrine cytokine loops, known to increase MM cell replication and de novo multidrug resistance. The second major adaptation was an increase in IL-6-independent growth rate, which correlates with the mutations observed in advanced stage patients. Even though there was an increase in the microvessel density in all simulations, the "angiogenic switch" was not due to a MM angiogenic phenotype, but rather the response of MM cells to the regional hypoxia caused by the increased tumor burden. These results indicate that treatments targeting the adaptation to survival and proliferation in hypoxia, in conjunction with currently available therapies, may have synergistic effects, by delaying tumor growth and reducing cytokine paracrine loops mediated by angiogenic factors.

Therapeutic Targeting of Protein Disulfide Isomerase PDIA1 in Multiple Myeloma.

Multiple myeloma is a genetically complex hematologic neoplasia in which malignant plasma cells constantly operate at the maximum limit of their unfolded protein response (UPR) due to a high secretory burden of immunoglobulins and cytokines. The endoplasmic reticulum (ER) resident protein disulfide isomerase, PDIA1 is indispensable for maintaining structural integrity of cysteine-rich antibodies and cytokines that require accurate intramolecular disulfide bond arrangement. PDIA1 expression analysis from RNA-seq of multiple myeloma patients demonstrated an inverse relationship with survival in relapsed or refractory disease, supporting its critical role in myeloma persistence. Using a structure-guided medicinal chemistry approach, we developed a potent, orally bioavailable small molecule PDIA1 inhibitor CCF642-34. The inhibition of PDIA1 overwhelms the UPR in myeloma cells, resulting in their apoptotic cell death at doses that do not affect the normal CD34+ hematopoietic stem and progenitor cells. Bortezomib resistance leads to increased PDIA1 expression and thus CCF642-34 sensitivity, suggesting that proteasome inhibitor resistance leads to PDIA1 dependence for proteostasis and survival. CCF642-34 induces acute unresolvable UPR in myeloma cells, and oral treatment increased survival of mice in the syngeneic 5TGM1 model of myeloma. Results support development of CCF642-34 to selectively target the plasma cell program and overcome the treatment-refractory state in myeloma.

IAP and HDAC inhibitors interact synergistically in myeloma cells through noncanonical NF-κB- and caspase-8-dependent mechanisms.

Interactions between the inhibitor of apoptosis protein antagonist LCL161 and the histone deacetylase inhibitor panobinostat (LBH589) were examined in human multiple myeloma (MM) cells. LCL161 and panobinostat interacted synergistically to induce apoptosis in diverse MM cell lines, including those resistant to bortezomib (PS-R). Similar interactions were observed with other histone deacetylase inhibitors (MS-275) or inhibitors of apoptosis protein antagonists (birinapant). These events were associated with downregulation of the noncanonical (but not the canonical) NF-κB pathway and activation of the extrinsic, caspase-8-related apoptotic cascade. Coexposure of MM cells to LCL161/LBH589 induced TRAF3 upregulation and led to TRAF2 and NIK downregulation, diminished expression of BCL-XL, and induction of γH2A.X. Ectopic expression of TRAF2, NIK, or BCL-XL, or short hairpin RNA TRAF3 knock-down, significantly reduced LCL161/LBH589 lethality, as did ectopic expression of dominant-negative FADD. Stromal/microenvironmental factors failed to diminish LCL161/LBH589-induced cell death. The LCL161/LBH589 regimen significantly increased cell killing in primary CD138+ cells (N = 31) and was particularly effective in diminishing the primitive progenitor cell-enriched CD138-/19+/20+/27+ population (N = 23) but was nontoxic to normal CD34+ cells. Finally, combined LCL161/LBH589 treatment significantly increased survival compared with single-agent treatment in an immunocompetent 5TGM1 murine MM model. Together, these findings argue that LCL161 interacts synergistically with LBH589 in MM cells through a process involving inactivation of the noncanonical NF-κB pathway and activation of the extrinsic apoptotic pathway, upregulation of TRAF3, and downregulation of TRAF2/BCL-XL. Notably, this regimen overcomes various forms of resistance, is active against primary MM cells, and displays significant in vivo activity. This strategy warrants further consideration in MM.

Transcriptional programming drives Ibrutinib-resistance evolution in mantle cell lymphoma.

Ibrutinib, a bruton's tyrosine kinase (BTK) inhibitor, provokes robust clinical responses in aggressive mantle cell lymphoma (MCL), yet many patients relapse with lethal Ibrutinib-resistant (IR) disease. Here, using genomic, chemical proteomic, and drug screen profiling, we report that enhancer remodeling-mediated transcriptional activation and adaptive signaling changes drive the aggressive phenotypes of IR. Accordingly, IR MCL cells are vulnerable to inhibitors of the transcriptional machinery and especially so to inhibitors of cyclin-dependent kinase 9 (CDK9), the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Further, CDK9 inhibition disables reprogrammed signaling circuits and prevents the emergence of IR in MCL. Finally, and importantly, we find that a robust and facile ex vivo image-based functional drug screening platform can predict clinical therapeutic responses of IR MCL and identify vulnerabilities that can be targeted to disable the evolution of IR.

Plasma cell dependence on histone/protein deacetylase 11 reveals a therapeutic target in multiple myeloma.

The clinical utility of histone/protein deacetylase (HDAC) inhibitors in combinatorial regimens with proteasome inhibitors for patients with relapsed and refractory multiple myeloma (MM) is often limited by excessive toxicity due to HDAC inhibitor promiscuity with multiple HDACs. Therefore, more selective inhibition minimizing off-target toxicity may increase the clinical effectiveness of HDAC inhibitors. We demonstrated that plasma cell development and survival are dependent upon HDAC11, suggesting this enzyme is a promising therapeutic target in MM. Mice lacking HDAC11 exhibited markedly decreased plasma cell numbers. Accordingly, in vitro plasma cell differentiation was arrested in B cells lacking functional HDAC11. Mechanistically, we showed that HDAC11 is involved in the deacetylation of IRF4 at lysine103. Further, targeting HDAC11 led to IRF4 hyperacetylation, resulting in impaired IRF4 nuclear localization and target promoter binding. Importantly, transient HDAC11 knockdown or treatment with elevenostat, an HDAC11-selective inhibitor, induced cell death in MM cell lines. Elevenostat produced similar anti-MM activity in vivo, improving survival among mice inoculated with 5TGM1 MM cells. Elevenostat demonstrated nanomolar ex vivo activity in 34 MM patient specimens and synergistic activity when combined with bortezomib. Collectively, our data indicated that HDAC11 regulates an essential pathway in plasma cell biology establishing its potential as an emerging theraputic vulnerability in MM.

A quantitative theoretical model for the development of malignancy in ductal carcinoma in situ.

Mathematical models and clinical observations have demonstrated that microenvironmental hypoxia and acidosis are important selection factors during the later stages of the somatic evolution of breast cancer. The consequent promotion of constitutive upregulation of glycolysis and resistance to acid-induced cellular toxicity is hypothesized to be critical for the ability of cancer cells to invade host tissue. In this work we developed a 3D fixed lattice cellular automata model to study the role of these two phenotypes in determining morphology and the potential for invasion of ductal carcinoma in situ (DCIS), which in this work is defined as the erosion of a healthy epithelial cell layer and direct contact with the basement membrane. The model was conceived as a 40-cell wide epithelial duct surrounded by blood vessels and composed of a basement membrane and one internal layer of epithelial cells. Our results show that an increment in the order of 8-fold in glucose metabolism and an increase in acid resistance corresponding to pH thresholds of approximately 6.8 and 6.45 for quiescence and death, respectively, are required for the tumor to breach through the layer of healthy epithelial cells and reach the basement membrane as a first step for invasion. Our model also suggests correlations between classic morphologies and different values of hyperglycolytic and acid-resistant phenotypes, indicating that immunohistochemistry studies targeting these genes may improve the predictive power of morphological analyses of biopsies.

Detection of clonal immunoglobulin and T-cell receptor gene rearrangements in childhood acute lymphoblastic leukemia using a low-cost PCR strategy.

BACKGROUND: Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD), which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL). We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method. Here, we report on gene rearrangement frequencies and offer guidelines for the application of the technique. PROCEDURE: Two hundred thirty-three children had DNA extracted from bone marrow. Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR. PCR products were submitted to homo/heteroduplex analysis. A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions. RESULTS: At least one clonal marker could be detected in 98% of the patients, and two markers in approximately 80%. The most commonly rearranged genes in precursor B-cell ALL were IgH (75%), TCRD (59%), IgK (55%), and TCRG (54%). The most commonly rearranged genes for T-ALL were TCRG (100%) and TCRD (24%). The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells. CONCLUSIONS: We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases. In addition, these reactions are suitable for MRD monitoring, especially when aiming the selection of patients with high MRD levels (≥ 10(-2)) at the end of induction therapy. Such an approach would be very useful in centers with limited financial resources.

A pharmacodynamic model of clinical synergy in multiple myeloma.

BACKGROUND: Multiagent therapies, due to their ability to delay or overcome resistance, are a hallmark of treatment in multiple myeloma (MM). The growing number of therapeutic options in MM requires high-throughput combination screening tools to better allocate treatment, and facilitate personalized therapy. METHODS: A second-order drug response model was employed to fit patient-specific ex vivo responses of 203 MM patients to single-agent models. A novel pharmacodynamic model, developed to account for two-way combination effects, was tested with 130 two-drug combinations. We have demonstrated that this model is sufficiently parameterized by single-agent and fixed-ratio combination responses, by validating model estimates with ex vivo combination responses for different concentration ratios, using a checkerboard assay. This new model reconciles ex vivo observations from both Loewe and BLISS synergy models, by accounting for the dimension of time, as opposed to focusing on arbitrary time-points or drug effect. Clinical outcomes of patients were simulated by coupling patient-specific drug combination models with pharmacokinetic data. FINDINGS: Combination screening showed 1 in 5 combinations (21.43% by LD50, 18.42% by AUC) were synergistic ex vivo with statistical significance (P < 0.05), but clinical synergy was predicted for only 1 in 10 combinations (8.69%), which was attributed to the role of pharmacokinetics and dosing schedules. INTERPRETATION: The proposed framework can inform clinical decisions from ex vivo observations, thus providing a path toward personalized therapy using combination regimens. FUNDING: This research was funded by the H. Lee Moffitt Cancer Center Physical Sciences in Oncology (PSOC) Grant (1U54CA193489-01A1) and by H. Lee Moffitt Cancer Center's Team Science Grant. This work has been supported in part by the PSOC Pilot Project Award (5U54CA193489-04), the Translational Research Core Facility at the H. Lee Moffitt Cancer Center & Research Institute, an NCI-designated Comprehensive Cancer Center (P30-CA076292), the Pentecost Family Foundation, and Miles for Moffitt Foundation.

BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models.

Drug-tolerant "persister" tumor cells underlie emergence of drug-resistant clones and contribute to relapse and disease progression. Here we report that resistance to the BCL-2 targeting drug ABT-199 in models of mantle cell lymphoma and double-hit lymphoma evolves from outgrowth of persister clones displaying loss of 18q21 amplicons that harbor BCL2. Further, persister status is generated via adaptive super-enhancer remodeling that reprograms transcription and offers opportunities for overcoming ABT-199 resistance. Notably, pharmacoproteomic and pharmacogenomic screens revealed that persisters are vulnerable to inhibition of the transcriptional machinery and especially to inhibition of cyclin-dependent kinase 7 (CDK7), which is essential for the transcriptional reprogramming that drives and sustains ABT-199 resistance. Thus, transcription-targeting agents offer new approaches to disable drug resistance in B-cell lymphomas.