Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

Acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL-2, creating a therapeutic opportunity to target LSCs using the BCL-2 inhibitor venetoclax. While venetoclax-based regimens have shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug-responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e. OXPHOS) status with relatively high levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in LSCs and provide an avenue for clinical management of venetoclax resistance.

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

We previously reported that acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL2, creating a therapeutic opportunity to target LSCs using the BCL2 inhibitor drug venetoclax. While venetoclax-based regimens have indeed shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence mechanisms that dictate venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e., OXPHOS) status with relatively high steady-state levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake sharply reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in the biology of LSCs and provide a therapeutic avenue for clinical management of venetoclax resistance.

Transcriptional adaptation of Mycobacterium tuberculosis that survives prolonged multi-drug treatment in mice.

To address the ongoing global tuberculosis crisis, there is a need for shorter, more effective treatments. A major reason why tuberculosis requires prolonged treatment is that, following a short initial phase of rapid killing, the residual Mycobacterium tuberculosis withstands drug killing. Because existing methods lack sensitivity to quantify low-abundance mycobacterial RNA in drug-treated animals, cellular adaptations of drug-exposed bacterial phenotypes in vivo remain poorly understood. Here, we used a novel RNA-seq method called SEARCH-TB to elucidate the Mycobacterium tuberculosis transcriptome in mice treated for up to 28 days with standard doses of isoniazid, rifampin, pyrazinamide, and ethambutol. We compared murine results with in vitro SEARCH-TB results during exposure to the same regimen. Treatment suppressed genes associated with growth, transcription, translation, synthesis of rRNA proteins, and immunogenic secretory peptides. Bacteria that survived prolonged treatment appeared to transition from ATP-maximizing respiration toward lower-efficiency pathways and showed modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pump expression. Although the pre-treatment in vivo and in vitro transcriptomes differed profoundly, genes differentially expressed following treatment in vivo and in vitro were similar, with differences likely attributable to immunity and drug pharmacokinetics in mice. These results reveal cellular adaptations of Mycobacterium tuberculosis that withstand prolonged drug exposure in vivo, demonstrating proof of concept that SEARCH-TB is a highly granular pharmacodynamic readout. The surprising finding that differential expression is concordant in vivo and in vitro suggests that insights from transcriptional analyses in vitro may translate to the mouse. IMPORTANCE A major reason that curing tuberculosis requires prolonged treatment is that drug exposure changes bacterial phenotypes. The physiologic adaptations of Mycobacterium tuberculosis that survive drug exposure in vivo have been obscure due to low sensitivity of existing methods in drug-treated animals. Using the novel SEARCH-TB RNA-seq platform, we elucidated Mycobacterium tuberculosis phenotypes in mice treated for with the global standard 4-drug regimen and compared them with the effect of the same regimen in vitro. This first view of the transcriptome of the minority Mycobacterium tuberculosis population that withstands treatment in vivo reveals adaptation of a broad range of cellular processes, including a shift in metabolism and cell wall modification. Surprisingly, the change in gene expression induced by treatment in vivo and in vitro was largely similar. This apparent "portability" from in vitro to the mouse provides important new context for in vitro transcriptional analyses that may support early preclinical drug evaluation.

Development and Multicenter Case-Control Validation of Urinary Comprehensive Genomic Profiling for Urothelial Carcinoma Diagnosis, Surveillance, and Risk-Prediction.

PURPOSE: Urinary comprehensive genomic profiling (uCGP) uses next-generation sequencing to identify mutations associated with urothelial carcinoma and has the potential to improve patient outcomes by noninvasively diagnosing disease, predicting grade and stage, and estimating recurrence risk. EXPERIMENTAL DESIGN: This is a multicenter case-control study using banked urine specimens collected from patients undergoing initial diagnosis/hematuria workup or urothelial carcinoma surveillance. A total of 581 samples were analyzed by uCGP: 333 for disease classification and grading algorithm development, and 248 for blinded validation. uCGP testing was done using the UroAmp platform, which identifies five classes of mutation: single-nucleotide variants, copy-number variants, small insertion-deletions, copy-neutral loss of heterozygosity, and aneuploidy. UroAmp algorithms predicting urothelial carcinoma tumor presence, grade, and recurrence risk were compared with cytology, cystoscopy, and pathology. RESULTS: uCGP algorithms had a validation sensitivity/specificity of 95%/90% for initial cancer diagnosis in patients with hematuria and demonstrated a negative predictive value (NPV) of 99%. A positive diagnostic likelihood ratio (DLR) of 9.2 and a negative DLR of 0.05 demonstrate the ability to risk-stratify patients presenting with hematuria. In surveillance patients, binary urothelial carcinoma classification demonstrated an NPV of 91%. uCGP recurrence-risk prediction significantly prognosticated future recurrence (hazard ratio, 6.2), whereas clinical risk factors did not. uCGP demonstrated positive predictive value (PPV) comparable with cytology (45% vs. 42%) with much higher sensitivity (79% vs. 25%). Finally, molecular grade predictions had a PPV of 88% and a specificity of 95%. CONCLUSIONS: uCGP enables noninvasive, accurate urothelial carcinoma diagnosis and risk stratification in both hematuria and urothelial carcinoma surveillance patients.

Transcriptional adaptation of drug-tolerant Mycobacterium tuberculosis in mice.

Transcriptome evaluation of Mycobacterium tuberculosis in the lungs of laboratory animals during long-term treatment has been limited by extremely low abundance of bacterial mRNA relative to eukaryotic RNA. Here we report a targeted amplification RNA sequencing method called SEARCH-TB. After confirming that SEARCH-TB recapitulates conventional RNA-seq in vitro, we applied SEARCH-TB to Mycobacterium tuberculosis-infected BALB/c mice treated for up to 28 days with the global standard isoniazid, rifampin, pyrazinamide, and ethambutol regimen. We compared results in mice with 8-day exposure to the same regimen in vitro. After treatment of mice for 28 days, SEARCH-TB suggested broad suppression of genes associated with bacterial growth, transcription, translation, synthesis of rRNA proteins and immunogenic secretory peptides. Adaptation of drug-stressed Mycobacterium tuberculosis appeared to include a metabolic transition from ATP-maximizing respiration towards lower-efficiency pathways, modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pumps expression. Despite markedly different expression at pre-treatment baseline, murine and in vitro samples had broadly similar transcriptional change during treatment. The differences observed likely indicate the importance of immunity and pharmacokinetics in the mouse. By elucidating the long-term effect of tuberculosis treatment on bacterial cellular processes in vivo, SEARCH-TB represents a highly granular pharmacodynamic monitoring tool with potential to enhance evaluation of new regimens and thereby accelerate progress towards a new generation of more effective tuberculosis treatment.

Urinary Comprehensive Genomic Profiling Correlates Urothelial Carcinoma Mutations with Clinical Risk and Efficacy of Intervention.

The clinical standard of care for urothelial carcinoma (UC) relies on invasive procedures with suboptimal performance. To enhance UC treatment, we developed a urinary comprehensive genomic profiling (uCGP) test, UroAmplitude, that measures mutations from tumor DNA present in urine. In this study, we performed a blinded, prospective validation of technical sensitivity and positive predictive value (PPV) using reference standards, and found at 1% allele frequency, mutation detection performs at 97.4% sensitivity and 80.4% PPV. We then prospectively compared the mutation profiles of urine-extracted DNA to those of matched tumor tissue to validate clinical performance. Here, we found tumor single-nucleotide variants were observed in the urine with a median concordance of 91.7% and uCGP revealed distinct patterns of genomic lesions enriched in low- and high-grade disease. Finally, we retrospectively explored longitudinal case studies to quantify residual disease following bladder-sparing treatments, and found uCGP detected residual disease in patients receiving bladder-sparing treatment and predicted recurrence and disease progression. These findings demonstrate the potential of the UroAmplitude platform to reliably identify and track mutations associated with UC at each stage of disease: diagnosis, treatment, and surveillance. Multiple case studies demonstrate utility for patient risk classification to guide both surgical and therapeutic interventions.

Identification of Zinc-Dependent Mechanisms Used by Group B Streptococcus To Overcome Calprotectin-Mediated Stress.

Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease.IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

Physiologic RNA targets and refined sequence specificity of coronavirus EndoU.

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for U ↓ A and C ↓ A sequences (endoY ↓ A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoUmut) or 2'-5' phosphodiesterase (PDEmut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U ↓ A and C ↓ A sequences (endoY ↓ A) within viral (+) strand RNA to evade dsRNA-activated host responses.

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries.

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1313 to 2002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.

High-Resolution Mapping of Modified DNA Nucleobases Using Excision Repair Enzymes.

Modification of DNA nucleobases has a profound effect on genome function. We developed a method that maps the positions of the modified DNA nucleobases throughout genomic DNA. This method couples in vitro nucleobase excision with massively parallel DNA sequencing to determine the location of modified DNA nucleobases with single base precision. This protocol was used to map uracil incorporation and UV photodimers in DNA, and a modification of the protocol has been used to map sparse modification events in cells. The Excision-seq protocol is broadly applicable to a variety of base modifications for which an excision enzyme is available.

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells.

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection.

High resolution mapping of modified DNA nucleobases using excision repair enzymes.

The incorporation and creation of modified nucleobases in DNA have profound effects on genome function. We describe methods for mapping positions and local content of modified DNA nucleobases in genomic DNA. We combined in vitro nucleobase excision with massively parallel DNA sequencing (Excision-seq) to determine the locations of modified nucleobases in genomic DNA. We applied the Excision-seq method to map uracil in E. coli and budding yeast and discovered significant variation in uracil content, wherein uracil is excluded from the earliest and latest replicating regions of the genome, possibly driven by changes in nucleotide pool composition. We also used Excision-seq to identify sites of pyrimidine dimer formation induced by UV light exposure, where the method could distinguish between sites of cyclobutane and 6-4 photoproduct formation. These UV mapping data enabled analysis of local sequence bias around pyrimidine dimers and suggested a preference for an adenosine downstream from 6-4 photoproducts. The Excision-seq method is broadly applicable for high precision, genome-wide mapping of modified nucleobases with cognate repair enzymes.

Chaperoning histones during DNA replication and repair.

Nuclear DNA is tightly packaged into chromatin, which profoundly influences DNA replication, transcription, repair, and recombination. The extensive interactions between the basic histone proteins and acidic DNA make the nucleosomal unit of chromatin a highly stable entity. For the cellular machinery to access the DNA, the chromatin must be unwound and the DNA cleared of histone proteins. Conversely, the DNA has to be repackaged into chromatin afterward. This review focuses on the roles of the histone chaperones in assembling and disassembling chromatin during the processes of DNA replication and repair.

FACT and the proteasome promote promoter chromatin disassembly and transcriptional initiation.

The packaging of the eukaryotic genome into chromatin represses gene expression by blocking access of the general transcription machinery to the underlying DNA sequences. Accordingly, eukaryotes have developed a variety of mechanisms to disrupt, alter, or disassemble nucleosomes from promoter regions and open reading frames to allow transcription to occur. Although we know that chromatin disassembly from the yeast PHO5 promoter is triggered by the Pho4 activator, the mechanism is far from clear. Here we show that the Pho4 activator can occupy its nucleosome-bound DNA binding site within the PHO5 promoter. In contrast to the role of Saccharomyces cerevisiae FACT (facilitates chromatin transcription) complex in assembling chromatin within open reading frames, we find that FACT is involved in the disassembly of histones H2A/H2B from the PHO5 promoter during transcriptional induction. We have also discovered that the proteasome is required for efficient chromatin disassembly and transcriptional induction from the PHO5 promoter. Mutants of the degradation function of the proteasome have a defect in recruitment of the Pho4 activator, whereas mutants of the ATPase cap of the proteasome do recruit Pho4 but are still delayed for chromatin assembly. Finally, we rule out the possibility that the proteasome or ATPase cap is driving chromatin disassembly via a potential ATP-dependent chromatin remodeling activity.

Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry.

The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.