The Proteostasis Network is a Therapeutic Target in Acute Myeloid Leukemia.

Oncogenic growth places great strain and dependence on the proteostasis network. This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for treatment of other cancers, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and autophagy, two key stem cell proteostasis pathways, to prevent unfolded protein accumulation. Inactivation of HSF1 sensitized human AML cells to proteasome inhibition, marked by unfolded protein accumulation, activation of the PERK-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival, and significant slowing of disease progression and extension of survival in vivo . Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed AML cells, and significantly reduced AML burden and extended survival in vivo . Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and induced apoptosis in primary patient AML cells, including AML stem/progenitor cells, without severely affecting normal hematopoietic stem/progenitor cells. Combined autophagy and proteasome inhibition also activated the integrated stress response, but surprisingly this occurred in a PKR-dependent manner. These studies unravel how proteostasis pathways are co-opted to promote AML growth, progression and drug resistance, and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.

Systemic deficits in lipid homeostasis promote aging-associated impairments in B cell progenitor development.

Organismal aging has been associated with diverse metabolic and functional changes across tissues. Within the immune system, key features of physiological hematopoietic cell aging include increased fat deposition in the bone marrow, impaired hematopoietic stem and progenitor cell (HSPC) function, and a propensity towards myeloid differentiation. This shift in lineage bias can lead to pre-malignant bone marrow conditions such as clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenias of undetermined significance (CCUS), frequently setting the stage for subsequent development of age-related cancers in myeloid or lymphoid lineages. At the systemic as well as sub-cellular level, human aging has also been associated with diverse lipid alterations, such as decreased phospholipid membrane fluidity that arises as a result of increased saturated fatty acid (FA) accumulation and a decay in n-3 polyunsaturated fatty acid (PUFA) species by the age of 80 years, however the extent to which impaired FA metabolism contributes to hematopoietic aging is less clear. Here, we performed comprehensive multi-omics analyses and uncovered a role for a key PUFA biosynthesis gene, ELOVL2 , in mouse and human immune cell aging. Whole transcriptome RNA-sequencing studies of bone marrow from aged Elovl2 mutant (enzyme-deficient) mice compared with age-matched controls revealed global down-regulation in lymphoid cell markers and expression of genes involved specifically in B cell development. Flow cytometric analyses of immune cell markers confirmed an aging-associated loss of B cell markers that was exacerbated in the bone marrow of Elovl2 mutant mice and unveiled CD79B, a vital molecular regulator of lymphoid progenitor development from the pro-B to pre-B cell stage, as a putative surface biomarker of accelerated immune aging. Complementary lipidomic studies extended these findings to reveal select alterations in lipid species in aged and Elovl2 mutant mouse bone marrow samples, suggesting significant changes in the biophysical properties of cellular membranes. Furthermore, single cell RNA-seq analysis of human HSPCs across the spectrum of human development and aging uncovered a rare subpopulation (<7%) of CD34 + HSPCs that expresses ELOVL2 in healthy adult bone marrow. This HSPC subset, along with CD79B -expressing lymphoid-committed cells, were almost completely absent in CD34 + cells isolated from elderly (>60 years old) bone marrow samples. Together, these findings uncover new roles for lipid metabolism enzymes in the molecular regulation of cellular aging and immune cell function in mouse and human hematopoiesis. In addition, because systemic loss of ELOVL2 enzymatic activity resulted in down-regulation of B cell genes that are also associated with lymphoproliferative neoplasms, this study sheds light on an intriguing metabolic pathway that could be leveraged in future studies as a novel therapeutic modality to target blood cancers or other age-related conditions involving the B cell lineage.

The Potential of JAG Ligands as Therapeutic Targets and Predictive Biomarkers in Multiple Myeloma.

The NOTCH ligands JAG1 and JAG2 have been correlated in vitro with multiple myeloma (MM) cell proliferation, drug resistance, self-renewal and a pathological crosstalk with the tumor microenvironment resulting in angiogenesis and osteoclastogenesis. These findings suggest that a therapeutic approach targeting JAG ligands might be helpful for the care of MM patients and lead us to explore the role of JAG1 and JAG2 in a MM in vivo model and primary patient samples. JAG1 and JAG2 protein expression represents a common feature in MM cell lines; therefore, we assessed their function through JAG1/2 conditional silencing in a MM xenograft model. We observed that JAG1 and JAG2 showed potential as therapeutic targets in MM, as their silencing resulted in a reduction in the tumor burden. Moreover, JAG1 and JAG2 protein expression in MM patients was positively correlated with the presence of MM cells in patients' bone marrow biopsies. Finally, taking advantage of the Multiple Myeloma Research Foundation (MMRF) CoMMpass global dataset, we showed that JAG2 gene expression level was a predictive biomarker associated with patients' overall survival and progression-free survival, independently from other main molecular or clinical features. Overall, these results strengthened the rationale for the development of a JAG1/2-tailored approach and the use of JAG2 as a predictive biomarker in MM.

Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells.

Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing factor mutations and mis-splicing fuel therapy-resistant leukemia stem cell (LSC) generation in adults, splicing deregulation has not been extensively studied in pAML. Herein, we describe single-cell proteogenomics analyses, transcriptome-wide analyses of FACS-purified hematopoietic stem and progenitor cells followed by differential splicing analyses, dual-fluorescence lentiviral splicing reporter assays, and the potential of a selective splicing modulator, Rebecsinib, in pAML. Using these methods, we discover transcriptomic splicing deregulation typified by differential exon usage. In addition, we discover downregulation of splicing regulator RBFOX2 and CD47 splice isoform upregulation. Importantly, splicing deregulation in pAML induces a therapeutic vulnerability to Rebecsinib in survival, self-renewal, and lentiviral splicing reporter assays. Taken together, the detection and targeting of splicing deregulation represent a potentially clinically tractable strategy for pAML therapy.

Reversal of malignant ADAR1 splice isoform switching with Rebecsinib.

Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However, inflammatory-microenvironment-induced ADAR1p110 to p150 splice isoform switching drives cancer stem cell (CSC) generation and therapeutic resistance in 20 malignancies. Previously, predicting and preventing ADAR1p150-mediated malignant RNA editing represented a significant challenge. Thus, we developed lentiviral ADAR1 and splicing reporters for non-invasive detection of splicing-mediated ADAR1 adenosine-to-inosine (A-to-I) RNA editing activation; a quantitative ADAR1p150 intracellular flow cytometric assay; a selective small-molecule inhibitor of splicing-mediated ADAR1 activation, Rebecsinib, which inhibits leukemia stem cell (LSC) self-renewal and prolongs humanized LSC mouse model survival at doses that spare normal hematopoietic stem and progenitor cells (HSPCs); and pre-IND studies showing favorable Rebecsinib toxicokinetic and pharmacodynamic (TK/PD) properties. Together, these results lay the foundation for developing Rebecsinib as a clinical ADAR1p150 antagonist aimed at obviating malignant microenvironment-driven LSC generation.

Proceedings from the Blood and Marrow Transplant Clinical Trials Network Myeloma Intergroup Workshop on Immune and Cellular Therapy in Multiple Myeloma.

The Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Myeloma Intergroup conducted a workshop on Immune and Cellular Therapy in Multiple Myeloma on January 7, 2022. This workshop included presentations by basic, translational, and clinical researchers with expertise in plasma cell dyscrasias. Four main topics were discussed: platforms for myeloma disease evaluation, insights into pathophysiology, therapeutic target and resistance mechanisms, and cellular therapy for multiple myeloma. Here we provide a comprehensive summary of these workshop presentations.

Sensitive intranuclear flow cytometric quantification of IRF4 protein in multiple myeloma and normal human hematopoietic cells.

Interferon regulatory factor 4 (IRF4) is a transcription factor that regulates normal and malignant immune cell development and is implicated in multiple myeloma pathogenesis. This protocol describes the use of combined cell surface and intranuclear staining with fluorescent antibodies to measure IRF4 protein expression within myeloma and normal immune cells. IRF4 protein quantification may provide a valuable prognostic tool to predict disease severity and sensitivity to IRF4-targeted therapies. This flow-cytometry-based procedure could also be rapidly translated into a clinically compatible assay. For complete details on the use and execution of this protocol, please refer to Mondala et al. (2021).

Selective antisense oligonucleotide inhibition of human IRF4 prevents malignant myeloma regeneration via cell cycle disruption.

In multiple myeloma, inflammatory and anti-viral pathways promote disease progression and cancer stem cell generation. Using diverse pre-clinical models, we investigated the role of interferon regulatory factor 4 (IRF4) in myeloma progenitor regeneration. In a patient-derived xenograft model that recapitulates IRF4 pathway activation in human myeloma, we test the effects of IRF4 antisense oligonucleotides (ASOs) and identify a lead agent for clinical development (ION251). IRF4 overexpression expands myeloma progenitors, while IRF4 ASOs impair myeloma cell survival and reduce IRF4 and c-MYC expression. IRF4 ASO monotherapy impedes tumor formation and myeloma dissemination in xenograft models, improving animal survival. Moreover, IRF4 ASOs eradicate myeloma progenitors and malignant plasma cells while sparing normal human hematopoietic stem cell development. Mechanistically, IRF4 inhibition disrupts cell cycle progression, downregulates stem cell and cell adhesion transcript expression, and promotes sensitivity to myeloma drugs. These findings will enable rapid clinical development of selective IRF4 inhibitors to prevent myeloma progenitor-driven relapse.

Alu-dependent RNA editing of GLI1 promotes malignant regeneration in multiple myeloma.

Despite novel therapies, relapse of multiple myeloma (MM) is virtually inevitable. Amplification of chromosome 1q, which harbors the inflammation-responsive RNA editase adenosine deaminase acting on RNA (ADAR)1 gene, occurs in 30-50% of MM patients and portends a poor prognosis. Since adenosine-to-inosine RNA editing has recently emerged as a driver of cancer progression, genomic amplification combined with inflammatory cytokine activation of ADAR1 could stimulate MM progression and therapeutic resistance. Here, we report that high ADAR1 RNA expression correlates with reduced patient survival rates in the MMRF CoMMpass data set. Expression of wild-type, but not mutant, ADAR1 enhances Alu-dependent editing and transcriptional activity of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist, and promotes immunomodulatory drug resistance in vitro. Finally, ADAR1 knockdown reduces regeneration of high-risk MM in serially transplantable patient-derived xenografts. These data demonstrate that ADAR1 promotes malignant regeneration of MM and if selectively inhibited may obviate progression and relapse.

RNA editing-dependent epitranscriptome diversity in cancer stem cells.

Cancer stem cells (CSCs) can regenerate all facets of a tumour as a result of their stem cell-like capacity to self-renew, survive and become dormant in protective microenvironments. CSCs evolve during tumour progression in a manner that conforms to Charles Darwin's principle of natural selection. Although somatic DNA mutations and epigenetic alterations promote evolution, post-transcriptional RNA modifications together with RNA binding protein activity (the 'epitranscriptome') might also contribute to clonal evolution through dynamic determination of RNA function and gene expression diversity in response to environmental stimuli. Deregulation of these epitranscriptomic events contributes to CSC generation and maintenance, which governs cancer progression and drug resistance. In this Review, we discuss the role of malignant RNA processing in CSC generation and maintenance, including mechanisms of RNA methylation, RNA editing and RNA splicing, and the functional consequences of their aberrant regulation in human malignancies. Finally, we highlight the potential of these events as novel CSC biomarkers as well as therapeutic targets.

RNA Splicing Modulation Selectively Impairs Leukemia Stem Cell Maintenance in Secondary Human AML.

Age-related human hematopoietic stem cell (HSC) exhaustion and myeloid-lineage skewing promote oncogenic transformation of hematopoietic progenitor cells into therapy-resistant leukemia stem cells (LSCs) in secondary acute myeloid leukemia (AML). While acquisition of clonal DNA mutations has been linked to increased rates of secondary AML for individuals older than 60 years, the contribution of RNA processing alterations to human hematopoietic stem and progenitor aging and LSC generation remains unclear. Comprehensive RNA sequencing and splice-isoform-specific PCR uncovered characteristic RNA splice isoform expression patterns that distinguished normal young and aged human stem and progenitor cells (HSPCs) from malignant myelodysplastic syndrome (MDS) and AML progenitors. In splicing reporter assays and pre-clinical patient-derived AML models, treatment with a pharmacologic splicing modulator, 17S-FD-895, reversed pro-survival splice isoform switching and significantly impaired LSC maintenance. Therapeutic splicing modulation, together with monitoring splice isoform biomarkers of healthy HSPC aging versus LSC generation, may be employed safely and effectively to prevent relapse, the leading cause of leukemia-related mortality.

Multiple myeloma-derived Jagged ligands increases autocrine and paracrine interleukin-6 expression in bone marrow niche.

Multiple myeloma cell growth relies on intrinsic aggressiveness, due to a high karyotypic instability, or on the support from bone marrow (BM) niche.We and other groups have provided evidences that Notch signaling is related to tumor cell growth, pharmacological resistance, localization/recirculation in the BM and bone disease.This study indicates that high gene expression levels of Notch signaling members (JAG1, NOTCH2, HES5 and HES6) correlate with malignant progression or high-risk disease, and Notch signaling may participate in myeloma progression by increasing the BM levels of interleukin-6 (IL-6), a major player in myeloma cell growth and survival. Indeed, in vitro results, confirmed by correlation analysis on gene expression profiles of myeloma patients and immunohistochemical studies, demonstrated that Notch signaling controls IL-6 gene expression in those myeloma cells capable of IL-6 autonomous production as well as in surrounding BM stromal cells. In both cases Notch signaling activation may be triggered by myeloma cell-derived Jagged ligands. The evidence that Notch signaling positively controls IL-6 in the myeloma-associated BM makes this pathway a key mediator of tumor-directed reprogramming of the bone niche.This work strengthens the rationale for a novel Notch-directed therapy in multiple myeloma based on the inhibition of Jagged ligands.

ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis.

Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1's effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.

An RNA editing fingerprint of cancer stem cell reprogramming.

BACKGROUND: Deregulation of RNA editing by adenosine deaminases acting on dsRNA (ADARs) has been implicated in the progression of diverse human cancers including hematopoietic malignancies such as chronic myeloid leukemia (CML). Inflammation-associated activation of ADAR1 occurs in leukemia stem cells specifically in the advanced, often drug-resistant stage of CML known as blast crisis. However, detection of cancer stem cell-associated RNA editing by RNA sequencing in these rare cell populations can be technically challenging, costly and requires PCR validation. The objectives of this study were to validate RNA editing of a subset of cancer stem cell-associated transcripts, and to develop a quantitative RNA editing fingerprint assay for rapid detection of aberrant RNA editing in human malignancies. METHODS: To facilitate quantification of cancer stem cell-associated RNA editing in exons and intronic or 3'UTR primate-specific Alu sequences using a sensitive, cost-effective method, we established an in vitro RNA editing model and developed a sensitive RNA editing fingerprint assay that employs a site-specific quantitative PCR (RESSq-PCR) strategy. This assay was validated in a stably-transduced human leukemia cell line, lentiviral-ADAR1 transduced primary hematopoietic stem and progenitor cells, and in primary human chronic myeloid leukemia stem cells. RESULTS: In lentiviral ADAR1-expressing cells, increased RNA editing of MDM2, APOBEC3D, GLI1 and AZIN1 transcripts was detected by RESSq-PCR with improved sensitivity over sequencing chromatogram analysis. This method accurately detected cancer stem cell-associated RNA editing in primary chronic myeloid leukemia samples, establishing a cancer stem cell-specific RNA editing fingerprint of leukemic transformation that will support clinical development of novel diagnostic tools to predict and prevent cancer progression. CONCLUSIONS: RNA editing quantification enables rapid detection of malignant progenitors signifying cancer progression and therapeutic resistance, and will aid future RNA editing inhibitor development efforts.

Neuroprotective effects of the anti-cancer drug sunitinib in models of HIV neurotoxicity suggests potential for the treatment of neurodegenerative disorders.

BACKGROUND AND PURPOSE: Anti-retrovirals have improved and extended the life expectancy of patients with HIV. However, as this population ages, the prevalence of cognitive changes is increasing. Aberrant activation of kinases, such as receptor tyrosine kinases (RTKs) and cyclin-dependent kinase 5 (CDK5), play a role in the mechanisms of HIV neurotoxicity. Inhibitors of CDK5, such as roscovitine, have neuroprotective effects; however, CNS penetration is low. Interestingly, tyrosine kinase inhibitors (TKIs) display some CDK inhibitory activity and ability to cross the blood-brain barrier. EXPERIMENTAL APPROACH: We screened a small group of known TKIs for a candidate with additional CDK5 inhibitory activity and tested the efficacy of the candidate in in vitro and in vivo models of HIV-gp120 neurotoxicity. KEY RESULTS: Among 12 different compounds, sunitinib inhibited CDK5 with an IC50 of 4.2 µM. In silico analysis revealed that, similarly to roscovitine, sunitinib fitted 6 of 10 features of the CDK5 pharmacophore. In a cell-based model, sunitinib reduced CDK5 phosphorylation (pCDK5), calpain-dependent p35/p25 conversion and protected neuronal cells from the toxic effects of gp120. In glial fibrillary acidic protein-gp120 transgenic (tg) mice, sunitinib reduced levels of pCDK5, p35/p25 and phosphorylated tau protein, along with amelioration of the neurodegenerative pathology. CONCLUSIONS AND IMPLICATIONS: Compounds such as sunitinib with dual kinase inhibitory activity could ameliorate the cognitive impairment associated with chronic HIV infection of the CNS. Moreover, repositioning existing low MW compounds holds promise for the treatment of patients with neurodegenerative disorders.

Correlations between commonly used objective signs and symptoms for the diagnosis of dry eye disease: clinical implications.

PURPOSE: To evaluate the relationship between signs and symptoms of dry eye disease (DED) in a clinic-based population. METHODS: In a retrospective analysis, clinical signs and symptoms were evaluated for 344 subjects (n = 82, normal; n = 263, dry eye), across 11 sites from the EU and United States. Pearson correlations between signs and symptoms (r(2) ) and an independent components analysis (ICA) mixing matrix were derived from the data set. Similar analysis was performed on an independent data set from 200 subjects in a previous study in Munich, Germany. RESULTS: No correlations above r(2) = 0.17 were found between any signs and symptoms, except for corneal and conjunctival staining, which reported an r(2) = 0.36. In the multisite study, the average r(2) for osmolarity (0.07), tear breakup time (0.12), Schirmer test (0.09), corneal (0.16) and conjunctival staining (0.17), meibomian grading (0.11) and Ocular Surface Disease Index(®) (0.11) were consistently low. Among patients who showed evidence of DED by consensus of clinical signs, only 57% reported symptoms consistent with a diagnosis of DED. Similar results were observed in the Munich-based study data set. Each component of the ICA mixing matrix exhibited minimal residual information. CONCLUSIONS: No consistent relationship was found between common signs and symptoms of DED. Each type of measurement provides distinct information about the condition of the ocular surface. These results also demonstrate that symptoms alone are insufficient for the diagnosis and management of DED and argue for a consensus of clinical signs that better reflect all aspects of the disease.

ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia.

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole-transcriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3ß implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.

Selective elimination of leukemia stem cells: hitting a moving target.

Despite the widespread use of chemotherapeutic cytotoxic agents that eradicate proliferating cell populations, patients suffering from a wide variety of malignancies continue to relapse as a consequence of resistance to standard therapies. In hematologic malignancies, leukemia stem cells (LSCs) represent a malignant reservoir of disease that is believed to drive relapse and resistance to chemotherapy and tyrosine kinase inhibitor (TKIs). Major research efforts in recent years have been aimed at identifying and characterizing the LSC population in leukemias, such as chronic myeloid leukemia (CML), which represents an important paradigm for understanding the molecular evolution of cancer. However, the precise molecular mechanisms that promote LSC-mediated therapeutic recalcitrance have remained elusive. It has become clear that the LSC population evolves during disease progression, thus presenting a serious challenge for development of effective therapeutic strategies. Multiple reports have demonstrated that LSC initiation and propagation occurs as a result of aberrant activation of pro-survival and self-renewal pathways regulated by stem-cell related signaling molecules including ß-catenin and Sonic Hedgehog (Shh). Enhanced survival in LSC protective microenvironments, such as the bone marrow niche, as well as acquired dormancy of cells in these niches, also contributes to LSC persistence. Key components of these cell-intrinsic and cell-extrinsic pathways provide novel potential targets for therapies aimed at eradicating this dynamic and therapeutically recalcitrant LSC population. Furthermore, combination strategies that exploit LSC have the potential to dramatically improve the quality and quantity of life for patients that are resistant to current therapies.

NOTCH1 signaling promotes human T-cell acute lymphoblastic leukemia initiating cell regeneration in supportive niches.

BACKGROUND: Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated. METHODOLOGY AND PRINCIPAL FINDINGS: We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34(+) cells from NOTCH1(Mutated) T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1(Wild-type) CD34(+) cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1(Mutated) CD34(+) fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1(Mutated) T-ALL LIC-engrafted mice and resulted in depletion of CD34(+)CD2(+)CD7(+) cells that harbor serial transplantation capacity. CONCLUSIONS: These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies.

Clinical utility of objective tests for dry eye disease: variability over time and implications for clinical trials and disease management.

PURPOSE: To evaluate the efficacy of commonly used biomarkers in dry eye disease management in a longitudinal observational case series study followed by an interventional study in a subset of subjects treated with cyclosporine A (0.05%). METHODS: Bilateral tear osmolarity, Schirmer, tear film breakup time (TBUT), staining, meibomian grading, and Ocular Surface Disease Index were measured for a period of 3 consecutive months in participants recruited from a clinic-based population at 2 study sites. Fifty-two subjects completed the study (n = 16 mild/moderate, n = 36 severe; age, 47.1 ± 16.1 years). After the 3-month observation period, severe dry eye patients were prescribed topical cyclosporine A and evaluated for an additional 3 months. RESULTS: Tear osmolarity (8.7 ± 6.3%) exhibited significantly less variability over a 3-month period than corneal staining (12.2 ± 8.8%, P = 0.040), conjunctival staining (14.8 ± 8.9%, P = 0.002), and meibomian grading (14.3 ± 8.8%, P < 0.0001) across the entire patient population. Osmolarity also demonstrated less variation than TBUT (11.7 ± 9.0%, P = 0.059), Schirmer tests (10.7 ± 9.2%, P = 0.67), and Ocular Surface Disease Index (9.3 ± 7.8%, P = 0.94), although the differences were not significant. Variation in osmolarity was less for mild dry eye patients (5.9 ± 3.1%) than severe dry eye patients (10.0 ± 6.9%, P = 0.038). After treatment, average osmolarity and variability were lowered from 341 ± 18 mOsm/L to 307 ± 8 mOsm/L (P < 0.0001, n = 10). A downward trend in symptoms followed changes in osmolarity, declining from 44 ± 17 mOsm/L to 38 ± 18 mOsm/L (P = 0.35). None of the other signs demonstrated a change after treatment. CONCLUSIONS: Over a 3-month period, tear film osmolarity was found to have the lowest variability among commonly used signs of dry eye disease. Reductions in osmolarity preceded changes in symptoms during therapy.