Search

Supplementary Figure 1 from BCR/ABL and Other Kinases from Chronic Myeloproliferative Disorders Stimulate Single-Strand Annealing, an Unfaithful DNA Double-Strand Break Repair

Supplementary Figure 1 from Targeting YB-1 in HER-2 Overexpressing Breast Cancer Cells Induces Apoptosis via the mTOR/STAT3 Pathway and Suppresses Tumor Growth in Mice

The Y-box binding protein-1 (YB-1) is a transcription/translation factor that is highly expressed in primary breast tumors where it is consistently associated with poor survival. It induces human epidermal growth factor receptor (her-2) along with its dimerization partner egfr by directly binding to their promoters. In addition to promoting growth by inducing receptor tyrosine kinases, YB-1 also protects cells against apoptosis through mechanisms that have not been fully revealed. Given this, we addressed whether YB-1 might be an eventual therapeutic target for breast cancer by inhibiting it with small interfering RNAs in vitro and in vivo. Inhibiting YB-1 suppressed the growth of six of seven breast cancer cell lines that had amplified her-2 or were triple negative. Importantly, targeting YB-1 induced apoptosis in BT474-m1 and Au565 breast cancer cells known to have her-2 amplifications. The potential role of signal transducers and activators of transcription 3 (STAT3) was pursued to address the underlying mechanism for YB-1–mediated survival. Inhibition of YB-1 decreased P-STAT3S727 but not P-STAT3Y705 or total STAT3. This was accompanied by decreased P-ERK1/2T202/Y204, P-mTORS2448, and total mammalian target of rapamycin mTOR. Furthering the role of STAT3 in these cells, we show that knocking it down recapitulated the induction of apoptosis. Alternatively, constitutively active P-STAT3 rescued YB-1–induced apoptosis. Finally, targeting YB-1 with 2 different siRNAs remarkably suppressed tumor cell growth in soft agar by >90% and delayed tumorigenesis in nude mice. We conclude that HER-2 overexpressing as well as triple-negative breast cancer cells are YB-1 dependent, suggesting it may be a good therapeutic target for these exceptionally aggressive tumors. [Cancer Res 2008;68(21):8661–6]

Supplementary Figure 2 from Targeting YB-1 in HER-2 Overexpressing Breast Cancer Cells Induces Apoptosis via the mTOR/STAT3 Pathway and Suppresses Tumor Growth in Mice

Supplementary Methods and Materials, Figures 1-6 from Y-Box Binding Protein-1 Induces the Expression of CD44 and CD49f Leading to Enhanced Self-Renewal, Mammosphere Growth, and Drug Resistance

Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that, collectively, maintain required outputs of mature blood cellsunder homeostatic conditions. An early developmental change in the HSC population in both species includes a postnatal switch from most of these cells existing in a rapidly cycling state and maintenance of a high self-renewal potential to a majority in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, if and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this gap, we isolated highly HSC-enriched CD34+CD38-CD45RA-CD90+CD49f+ ("CD49f+") cells from cord blood and adult bone marrow and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth-factor stimulation in vitro and regenerative activity in vivo in transplanted mice. Initial experiments revealed advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth-factor stimulated proliferation of CD49f+ cells mediated by reduced intrinsic activation of AKT and altered cell-cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive normal human hematopoietic cells.

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRASG12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.

Despite the effectiveness of tyrosine kinase inhibitors (TKIs) against chronic myeloid leukemia (CML), they are not usually curative as some patients develop drug-resistance or are at risk of disease relapse when treatment is discontinued. Studies have demonstrated that primitive CML cells display unique miRNA profiles in response to TKI treatment. However, the utility of miRNAs in predicting treatment response is not yet conclusive. Here, we analyzed differentially expressed miRNAs in CD34

Cancer metabolism adapts the metabolic network of its tissue of origin. However, breast cancer is not a disease of a single origin. Multiple epithelial populations serve as the culprit cell of origin for specific breast cancer subtypes, yet our knowledge of the metabolic network of normal mammary epithelial cells is limited. Using a multi-omic approach, here we identify the diverse metabolic programmes operating in normal mammary populations. The proteomes of basal, luminal progenitor and mature luminal cell populations revealed enrichment of glycolysis in basal cells and of oxidative phosphorylation in luminal progenitors. Single-cell transcriptomes corroborated lineage-specific metabolic identities and additional intra-lineage heterogeneity. Mitochondrial form and function differed across lineages, with clonogenicity correlating with mitochondrial activity. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed lineage-rooted metabolic vulnerabilities of mammary progenitors. Bioinformatics indicated breast cancer subtypes retain metabolic features of their putative cell of origin. Thus, lineage-rooted metabolic identities of normal mammary cells may underlie breast cancer metabolic heterogeneity and targeting these vulnerabilities could advance breast cancer therapy.

Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of microRNAs (miRNAs) in regulating drug resistance and leukemic stem cell (LSC) fate, we performed global transcriptome profiling in treatment-naive chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor: its restored expression impaired survival of drug-resistant cells, sensitized them to TKIs in vitro, and markedly eliminated long-term repopulating LSCs and infiltrating blast cells, conferring a survival advantage in preclinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185, and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity, sensitizing therapy-resistant cells to TKIs. Thus, miR-185 presents as a potential predictive biomarker, and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL1 may provide a valuable strategy for overcoming drug resistance in patients.

Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a–null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a−/− myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a−/− HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a−/− mice. miR-146a−/− HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

The magnitude and scope of intrinsic age-correlated and host endocrine age-correlated gene expression in breast cancer is not well understood. From age-correlated gene expression in 3,071 breast cancer transcriptomes and epithelial protein expression of 42 markers in 5,001 breast cancers and 537 normal breast tissues, we identified a majority of age-correlated genes as putatively regulated by age-dependent estrogen signaling. Surprisingly, these included genes encoding the chromatin modifier EZH2 (which had a negative age correlation) and associated H3K27me3 (which had an inverse, positive age correlation). Among The Cancer Genome Atlas lung, thyroid, kidney and prostate transcriptomes, the largest overlap with breast cancer in age-correlated transcripts was lung cancer, for which about one-third of overlapping age-correlated transcripts appeared to be estrogen regulated. Age-quartile-stratified outcomes analysis of 3,500 breast cancers using EZH2, H3K27me3, FOXA1 and BCL2 proteins revealed distinct age-related prognostic significance. Age correlation in gene expression may thus be an important factor in ER, EZH2, H3K27me3 and other biomarker assessment and treatment strategies. Aparicio and colleagues identify gene expression changes in breast cancer datasets putatively associated with age-related endocrine effects, suggesting that patient age may influence the prognostic potential of certain biomarkers.

A problematic feature of many human cancers is a lack of understanding of mechanisms controlling organ-specific patterns of metastasis, despite recent progress in identifying many mutations and transcriptional programs shown to confer this potential. To address this gap, we developed a methodology that enables different aspects of the metastatic process to be comprehensively characterized at a clonal resolution. Our approach exploits the application of a computational pipeline to analyze and visualize clonal data obtained from transplant experiments in which a cellular DNA barcoding strategy is used to distinguish the separate clonal contributions of two or more competing cell populations. To illustrate the power of this methodology, we demonstrate its ability to discriminate the metastatic behavior in immunodeficient mice of a well-established human metastatic cancer cell line and its co-transplanted LRRC15 knockdown derivative. We also show how the use of machine learning to quantify clone-initiating cell (CIC) numbers and their subsequent metastatic progeny generated in different sites can reveal previously unknown relationships between different cellular genotypes and their initial sites of implantation with their subsequent respective dissemination patterns. These findings underscore the potential of such combined genomic and computational methodologies to identify new clonally-relevant drivers of site-specific patterns of metastasis.

Caspase-3 is a widely expressed member of a conserved family of proteins, generally recognized for their activated proteolytic roles in the execution of apoptosis in cells responding to specific extrinsic or intrinsic inducers of this mode of cell death. However, accumulating evidence indicates that caspase-3 also plays key roles in regulating the growth and homeostatic maintenance of both normal and malignant cells and tissues in multicellular organisms. Given that yeast possess an ancestral caspase-like gene suggests that the caspase-3 protein may have acquired different functions later during evolution to better meet the needs of more complex multicellular organisms, but without necessarily losing all of the functions of its ancestral yeast precursor. This review provides an update on what has been learned about these interesting dichotomous roles of caspase-3, their evolution, and their potential relevance to malignant as well as normal cell biology.

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins (FPs) are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of acute myeloid leukemia (AML) driven by the most common AF10 FPs, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent antioncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.

High YAP activity is associated with poor prognosis human breast cancers, but its role during the initial stage of mammary cell transformation is unknown. To address this question, we designed experiments that exploit the ability of KRASG12D-transduced subsets of freshly isolated normal human mammary cells to form invasive tumors rapidly and efficiently when transplanted into immunodeficient mice. Initial examination of the newly developing tumors thus generated revealed a consistent marked loss of nuclear YAP, independent of the initial primary human mammary cell type transduced. Conversely, co-transduction of the same subsets of primary human mammary cells with KRASG12D plus the constitutively active YAPS127A prevented tumor formation. These findings contrast with the enhanced display of transformed properties obtained when the immortalized, but non-tumorigenic MCF10A cells are transduced just with YAPS127A. In addition, we show that YAPS127A-transduction of the human MDA-MB-231 breast cancer cell line (that carry a similar KRAS mutation) enhances their metastatic activity in vivo. We also discover that the KRASG12D-induced early loss of YAP in primary human mammary cells is associated with their induced secretion of amphiregulin. Collectively, these findings suggest that YAP can differentially affect the acquisition of malignant properties by human mammary cells at different stages of their transformation.

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

The telomeric amplicon at 8p12 is common in oestrogen receptor-positive (ER+) breast cancers. Array-CGH and expression analyses of 1172 primary breast tumours revealed that ZNF703 was the single gene within the minimal amplicon and was amplified predominantly in the Luminal B subtype. Amplification was shown to correlate with increased gene and protein expression and was associated with a distinct expression signature and poor clinical outcome. ZNF703 transformed NIH 3T3 fibroblasts, behaving as a classical oncogene, and regulated proliferation in human luminal breast cancer cell lines and immortalized human mammary epithelial cells. Manipulation of ZNF703 expression in the luminal MCF7 cell line modified the effects of TGFβ on proliferation. Overexpression of ZNF703 in normal human breast epithelial cells enhanced the frequency of in vitro colony-forming cells from luminal progenitors. Taken together, these data strongly point to ZNF703 as a novel oncogene in Luminal B breast cancer.

The ability to use the Long-Term Bone Marrow Culture system as a quantitative assay for a primitive hemopoietic cell should allow the future assessment of the frequency of this cell in the blood and bone marrow of patients with diseases affecting hemopoiesis, or after chemotherapy or other in vivo treatments. Colony assays have also proven to be of routine utility in the diagnosis and assessment of patients with primary hematologic disorders or with secondarily perturbed hemopoiesis. The ability to observe and manipulate the differentiative behavior of these primitive hemopoietic cells in vitro has provided a powerful tool for the investigation of both normal and abnormal hemopoiesis. Pure megakaryocyte colonies are usually defined on the basis of the presence of at least two megakaryocytes, which, because of their resemblance to macrophages in unstained preparations, are best identified by positive staining for lineage-specific markers.

Assessment of autophagy activity has historically been limited to investigations of fixed tissue or bulk cell populations. To address questions of heterogeneity and relate measurements to functional properties of viable cells isolated from primary tissue, we created a lentiviral (RFP-GFP-MAP1LC3B) vector that allows the autophagosome and autolysosome content of transduced cells to be monitored at the single-cell level. Use of this strategy to analyze purified subsets of normal human mammary cells showed that both the luminal progenitor-containing (LP) subset and the basal cells (BCs) display highly variable but overall similar autophagic flux activity despite differences suggested by measurements of the proteins responsible (i.e., LC3B, ATG7 and BECLIN1) in bulk lysates. Autophagosome content was also highly variable in the clonogenic cells within both the LPs and BCs, but the proliferative response of the BCs was more sensitive to autophagy inhibition. In addition, use of this vector showed cells with the lowest autophagosome content elicited the fastest tumor growth in 2 different models of human mammary tumorigenesis. These results illustrate the utility of this vector to define differences in the autophagy properties of individual cells in primary tissue and couple these with their responses to proliferative and oncogenic stimuli.

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of AML driven by the most common AF10 fusion proteins, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a comprehensive map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent anti-oncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.STATEMENT OF SIGNIFICANCEGene fusions of AF10/MLLT10 are recurrent in acute myeloid and lymphoid leukemia and are associated with extremely poor survival outcomes. We show that the JAK1 kinase is required for activation of the AF10 fusion oncotranscriptome and for leukemogenesis. Since a number of JAK/STAT pathways inhibitors are in clinical development or approved for use, our studies may help develop a therapeutic strategy for AF10-rearranged leukemias.

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38−) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.

BACKGROUND: Human prostate cancers display numerous DNA methylation changes compared to normal tissue samples. However, definitive identification of features related to the cells' malignant status has been compromised by the predominance of cells with luminal features in prostate cancers. METHODS: We generated genome-wide DNA methylation profiles of cell subpopulations with basal or luminal features isolated from matched prostate cancer and normal tissue samples. RESULTS: Many frequent DNA methylation changes previously attributed to prostate cancers are here identified as differences between luminal and basal cells in both normal and cancer samples. We also identified changes unique to each of the two cancer subpopulations. Those specific to cancer luminal cells were associated with regulation of metabolic processes, cell proliferation and epithelial development. Within the prostate cancer TCGA dataset, these changes were able to distinguish not only cancers from normal samples, but also organ-confined cancers from those with extraprostatic extensions. Using changes present in both basal and luminal cancer cells, we derived a new 17-CpG prostate cancer signature with high predictive power in the TCGA dataset. CONCLUSIONS: This study demonstrates the importance of comparing phenotypically matched prostate cell populations from normal and cancer tissues to unmask biologically and clinically relevant DNA methylation changes.

Characterization of human cells that sustain blood cell production lifelong has historically been inferred from phenotypically defined subsets of cells assayed in vitro, in transplanted immunodeficient mice, or in patients transplanted with genetically marked cells. These approaches have led to the concept of a persistent complex hierarchical process of differentiation divisions originating from a rare population of CD34+CD38−CD45RA−CD90+CD49f+ cells with an average self-renewal potential of >0.5 and an ability to produce some or all blood cell types for >1 year. However, the role of these “49f” cells in the unperturbed adult has remained poorly understood. To address this gap, somatic single-nucleotide polymorphisms (SNVs) have recently been exploited as lineage tracing markers to enumerate and characterize active hematopoietic clones in normal adults using a capture and recapture approach. We show here that the use of somatic transversions to identify somatically acquired variant alleles enabled their detection in bulk populations at frequencies of approximately 1 in 80,000 cells. We then applied this method to blood cells isolated from two normal adults (aged 31 and 53 years) over a 1- to 3-year period. The results revealed in both donors a continued clonal output of both T- and B-lymphoid cells as well as myeloid cells identified by the same unique transversions found to distinguish single 49f cells isolated from the same donors’ initial blood samples. These findings provide the first evidence of a continuing hematopoietic stem cell–derived source of all mature blood cell types in normal (unperturbed) adult humans.

TO THE EDITOR: The recent Blood article by Schroeder et al demonstrates that plerixafor mobilizes a unique hematopoietic stem and progenitor cell (HSPC) product that is enriched in plasmacytoid dendritic cell (pDC) precursors (pre-pDCs).[1][1] This study further reports that enrichment of these

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.

This abstract was not presented at the conference. Citation Format: Chatterjee S, Basak P, Tan S, Lefort S, Pellacani D, Safneck J, Buchel E, Aparicio S, Eaves CJ, Raouf A. Not presented [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-04.

Lifelong production of most types of mature blood cells is sustained by a small population of cells with extensive regenerative potential. However, the detailed steps that restrict multipotent or even bipotent human hematopoietic cells to any single lineage remain poorly understood. Those that segregate the human B-lymphoid and neutrophil/monocyte (NM) lineages are of particular interest as these appear to identify a stage that might control the different properties of human leukemias that display perturbed NM and/or B-cell programs. To undertake a refined analysis of this normal lineage restriction process in human cells, we first devised a culture system that permits it to be tracked clonally and efficiently (50%). Initial experiments showed this could be achieved using a combination of multiple stromal cell types and human growth factor-supplemented medium. To elucidate the intervening transitional steps we then used multiplexed flow cytometry to compare the progeny generated in this culture system over a 2-week time course from previously defined lymphoid progenitor-enriched (P-L), NM progenitor-enriched (P-NM) and less restricted P-mix cord blood (CB) subsets. The results suggested that gain of CD45RA (RA) expression and loss of CLEC12A (C) expression appeared to accompany the sequential restriction of early CD34+ progenitors first to cells with dual NM+B-lineage potential and then just to B-lineage potential. Subsequent tracking of the lineage outputs of CD34+ RA-C- cells initially produced in larger numbers from unfractionated CD34+ CB cells either in vitro or in xenografted immunodeficient mice, confirmed the CD34+ RA-C- subset to be highly enriched in cells with dual NM+B potential. In contrast, co-generated CD45RA+ (RA+C-) and RA+CLEC12A+ (RA+C+) phenotypes displayed separate B- and NM lineage-restricted activity, respectively, as indicated by their largely exclusive clonal outputs of CD19+ pre-B and CD14+/CD15+ NM precursors. In agreement with these phenotypically established separate NM and B-lineage outputs, RA+C- cells were found to contain higher levels of B-lineage-associated gene transcripts (e.g., DNTT, CD79A, and EBF1), whereas RA+C+ cells contained higher levels of the NM transcription factor mRNAs encoded by SPI1 and CEBPA. In a further optimized stroma-free liquid culture system, the RA-C- cells could be shown to produce continuously RA+C- and RA+C+ progeny after another 3-4 days, and also RA-C- progeny which are not produced from more restricted RA+C- and RA+C+ cells, suggesting that the acquired expression of RA precedes the separation of NM and B-lineage potential that is then marked by the differential activation of C expression in RA+ cells. To examine more precisely how the process of B+NM restriction to a single lineage might be related to successive cell cycles, we labeled RA-C- cells with carboxyfluorescein diacetate succinimidyl ester (CFSE) to enable the phenotypes and growth potential of the the progeny obtained after 6 days to be directly related to their prior division histories. This revealed extensive heterogeneity in the overall distribution of the initial progeny cell cycle times but with a clear segregation in the outputs of the faster and slower dividing cells. Notably, the faster dividers produced ultimately small M+B or uni-lineage clones whereas the initially slow dividers subsequently produced larger clones, 25% of which still contained CD34+ cells or N+M+B progeny. Taken together, these findings identify new hallmark phenotypic changes that identify a critical step in the lineage restriction of primitive human hematopoietic cells with dual NM+B-lineage potential and a previously unknown association of this process with a shortening of their cell cycle transit time. Disclosures No relevant conflicts of interest to declare.

Caspase-3 has long been recognized for its key role as a protease that mediates the degradation of cellular components during the late stages of apoptosis. However, there is also a growing body of evidence that caspase-3 has essential intrinsic functions in regulating normal and malignant cell properties that are distinct from its apoptotic functions. In a previous study, we showed that a readily detectable but low proportion of freshly isolated normal human mammary cells with a luminal phenotype and a capacity to divide in vitro display a reversible expression of activated caspase-3, and subsequently it was found that suppression of caspase-3 expression in the MDA-MB-231 human breast cancer cell line contributes to its radiation-induced loss of proliferative activity in vitro. To further investigate the role of caspase-3 in normal and malignant human mammary cells, we constructed shRNA-caspase-3 and control lentiviral vectors to examine the effect of decreasing caspase-3 expression (2-fold determined by Western blot) in normal and transformed human mammary cell types. Knockdown (KD) of caspase-3 expression in 2 breast cancer cell lines (MDA-MB-231 and MCF7 cells) showed that caspase-3 KD suppressed their growth in vitro 5-fold within 8 days and inhibited formation of tumors in highly immunodeficient (NRG) adult female mice transplanted subcutaneously with shCaspase-3-transduced cells in contrast to controls in which 1cm diameter tumors appeared within 6 weeks. The shCaspase-3-transduced cells also showed a complete loss of lung metastasizing activity in intravenously injected mice. A similar anti-tumorigenic effect was also obtained in freshly isolated normal human mammary cells that were co-transduced with the shCaspase-3 vector as well as KRASG12D which otherwise causes the normal cells to produce invasive ductal carcinomas in subcutaneously transplanted immunodeficient mice. Annex V and propidium iodide staining of single-cell, as well as bulk cultures of the MDA-MB-231cells, showed that Caspase-3 is required for their survival as well as their growth. Surprisingly, initial parallel experiments with purified basal and luminal progenitor subsets of normal human mammary cells and the immortalized but non-tumorigenic MCF-10A cell line also indicates a significant suppression of cell growth in vitro within 10 days. These findings highlight the multiplicity of pro- as well as anti-survival activities that Caspase-3 exerts on normal and malignant human mammary cells raising important considerations of any therapy designed to augment their activity. Keywords: Breast cancer, Caspase-3, Cell survival Citation Format: Ebrahim Eskandari, Connie J. Eaves, Susanna Tan. Caspase 3 plays a requisite role in regulating survival of human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1949.

Human breast cancers are genetically and biologically heterogeneous with variable responses to current treatments. As an alternative strategy to identify causal and shared mechanisms, we have been exploring the utility of creating de novo models of human breast cancers from freshly isolated, purified subsets of normal human mammary cells that are lentivirally transduced with defined genes of interest and then transplanted subcutaneously in Matrigel into highly immunodeficient adult female Nonobese-diabetic/Rag1-/--IL2Rγc-/- (NRG) mice. In an initial study using this approach, we discovered that forced expression of KRASG12D alone rapidly generates small (growth-arrested), but polyclonal and serially transplantable invasive ductal carcinomas (IDCs) from both normal basal and luminal progenitor subsets of normal human mammary cells. This outcome is neither replaced nor influenced by transduction of the cells with mutant forms of TP53 or PI3KCA (Nguyen et al Nature 2015). More recently, we have found that transduction of these normal human mammary cells with a cDNA encoding a myristylated form of AKT1 (myrAKT1) produces a model of ductal carcinoma in situ (DCIS), whereas forced expression of BMI1, MYC and TP53R273C with KRASG12D (KBMT) rapidly produces large and aggressive tumors within 6 weeks post-transplant, with intermediate outcomes obtained when either MYC or TP53R273C is omitted. Immunohistochemical staining for estrogen, progesterone and epidermal growth factor receptor 2 suggests that the KBMT tumors are triple negative, and that the proportion of proliferating cells in the KBMT tumors is higher than in the tumors induced by just KRASG12D. The latter finding is consistent with the known role of BMI1 as a positive upstream regulator of proliferation via its ability to inhibit expression of p16INK4a, although the relevance of this activity in the KBMT model has yet to be established. At the opposite end of the spectrum, we have also shown that co-induced suppression of Y-Box binding protein-1 (YBX1) expression blocks the in vivo growth of KRASG12D-induced IDCs and its forced increased expression enhances the growth of myrAKT-induced DCIS cells. Taken together, these findings illustrate the advantages of creating and characterizing de novo models of human breast cancers to identify and manipulate pathways that are required for the acquisition of malignant properties. Citation Format: Susanna Tan, Sylvain Lefort, Amal El-Naggar, Davide Pellacani, Poul H. Sorensen, Connie Eaves. First report of a full spectrum of de novo induced human breast cancer subtypes generated from oncogene-transduced freshly isolated normal human mammary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 125.

Summary: The normal adult human female mammary gland is a bilayered structure consisting of an outer basal layer and two readily distinguished subsets of cells within the inner luminal layer. We now present a validated methodology for undertaking large-scale multi-parameter mass cytometric analyses of these cell types at single-cell resolution. In addition, we show how combining this approach with in vitro clonogenic assays of the proliferative and signaling responses of normal human mammary cells to epidermal growth factor (EGF) allows additional subsets with different EGF responses to be discerned. This included the identification of a subset of cells within the phenotypically defined luminal progenitor fraction that displays an elevated content of active caspase-3, including some that generate clones in vitro in response to EGF, with immunohistochemical evidence of their presence in situ in fixed preparations of normal human breast tissue. : Knapp et al. identify a subpopulation of progenitors in the human mammary gland that have partially activated an apoptotic program despite retaining some viability and clonogenic potential. As such activation has been linked to genomic instability, these cells may be at increased risk for oncogenic transformation. Keywords: mammary, mass cytometry, cancer, apoptosis, signaling, cell death, genomic instability, single-cell biology, growth factors