A Whole Genome In Vivo Crispr Screen in Primary ALL Predicts Leukaemic Relapse

Abstract Although paediatric acute lymphoblastic leukaemia (ALL) has a favourable prognosis, a number of cases will invariably relapse. One of the major problems associated with relapse is drug resistance, in particular to glucocorticoids, the mainstay of ALL treatment. Examining the underlying mechanisms is complicated by clonal heterogeneity within a patient and the potential impact of the leukaemic niche. To address mechanisms of drug resistance in a patient-relevant setting, we performed a genome-wide in vivo CRISPR screen in primary ALL material. To that end, we took advantage of primografted material from patient L707, who initially presented with a Dexamethasone (DEX) sensitive t(17;19) ALL, but relapsed 5 months after initial diagnosis. We transduced DEX sensitive presentation cells with the full genome GeCKOv2 CRISPR library, before transplantation into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were subsequently treated with DEX by oral gavage (15mg/kg for 5 weeks, 10mg/kg thereafter). DNA from several engrafted sites in the mouse was extracted and PCR amplified before being sequenced on the Illumina HiSeq2500. Changes in pool complexity were analysed using MaGEcK software to determine which sgRNAs were significantly enriched or depleted. By far the most significantly enriched sgRNAs were those targeting NR3C1, the gene encoding the glucocorticoid receptor. In addition, two of the top five significantly depleted sgRNAs targeted the Plexins, PLXNA1 and PLXND1. Whilst PLXNA1 is expressed at low levels, PLXND1 is highly expressed and has been linked to dexamethasone resistance. Notably, the matched relapsed material from L707 was highly DEX resistant both in tissue culture and when transplanted into NSG mice. SNP 6.0 analysis revealed a 5q deletion in the relapse, spanning 5 genes including NR3C1. Whole genome sequencing showed this was comprised of 2 deletions both targeting NR3C1, with different breakpoints for each allele. The differential gene expression between the L707 presentation and relapse established that NR3C1 was the most significant of all the genes lost at relapse, based on gene set enrichment analysis (GSEA). This contrasts with many ALL cases, where one of the downstream effectors of apoptosis is lost as opposed to NR3C1. Growth of the relapse material in vivo and in vitro was slower than the presentation in a competitive situation, but with DEX treatment the relapse phenotype began to emerge with a small percentage of cells showing a heterozygous deletion of NR3C1. These combined data strongly suggest that the NR3C1 deletion is the main driver of DEX resistance in the L707 relapse. Moreover, it proves that our in vivo CRISPR screen predicted the leukaemic relapse. These results confirm NR3C1 deletion as a driver in glucocorticoid resistance and demonstrate the power of in vivo CRISPR screens to predict mechanisms of gain of drug resistance and subsequent relapse. The parallels that can be drawn between the relapse and the CRISPR screen are striking, giving the indication that the progression from presentation to relapse may follow the same path in a patient derived xenograft setting as it did in the patient. Disclosures No relevant conflicts of interest to declare.

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Enhancing Functional Platelet Release In Vivo from in Vitro-Grown Megakaryocytes Using the Protein Kinase Inhibitor SU6656

Blood ◽

10.1182/blood.v128.22.1029.1029 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1029-1029

Author(s):

Danuta Jadwiga Jarocha ◽

Karen K Vo ◽

Randolph B Lyde ◽

Vincent M Hayes ◽

Mortimer Poncz

Keyword(s):

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Specific Inhibitor ◽

Annexin V ◽

Rock Inhibitor ◽

Medicine Research ◽

Nsg Mice ◽

Platelet Release

Abstract The clinical demand for platelet transfusions is increasing, threatening the ability to obtain sufficient healthy donors to provide these platelets. Advances in regenerative medicine research have opened the possibility of generating sufficient in vitro-grown megakaryocytes and consequent platelets to supply a portion of the clinical platelet transfusion demand. We have shown that infusing megakaryocytes for obtaining released, functional platelets is a viable alternative strategy than trying to release platelets in vitro. However, for both approaches, in vitro-cultured megakaryocytes have lower ploidy and release fewer platelets than likely occurs in vivo by primary cells. SU6656 inhibitor, a Src kinase inhibitor, has been shown to influence ploidization in several megakaryocyte-like line with purported increase in proplatelets release. However, in our hands, other agents - such as the ROCK inhibitor Y27632 - while increasing polyploidization markedly, inhibited platelet release per infused megakaryocyte in vivo. We grew megakaryocytes from CD34+ cells for 12 days with or without SU6656 (2.5 µM) supplementation during the last 4 days. We found that the SU6656 inhibitor only increased the number of CD34+-derived megakaryocytes by ~15% at the end of the 12 day growth, but more markedly increase the percent of large megakaryocytes measured by FSC parameter in flow cytometry evaluation from 28 up to 41% and percent of high granular megakaryocytes from 27 to 45%. These changes were accompanied with a shift in average ploidy from 4.9 to 6.9 (p<0.0003, N=6). Notably, SU6656-treated megakaryocytes released ~4-fold more platelets per infused megakaryocytes in immunocompromized NSG mice than untreated similarly in vitro-grown megakaryocytes. By 24 hrs, there were 6.5-fold platelets from the infused SU6656-treated megakaryocytes than control untreated (p<0.037, N=6). Released platelets from the drug-treated and untreated megakaryocytes had similar levels of percent thiazole orange positivity as an indication that they were young platelets. Importantly, baseline annexin V, CD62p and PAC1 binding prior to agonist exposure were also similarly and increased to the same extent after thrombin (1U/ml) stimulation. Additionally, incorporation into a growing cremaster laser injury-induced thrombus in vivo was similar further indicating retained function by the platelets released from the drug-treated megakaryocytes. A number of strategies such as modifying the level of transcription factors have been proposed to increase the size, ploidy or proplatelets release from in vitro-grown megakaryocytes. In none of these cases have these released platelets in vivo biology been examined and demonstrated to replicate high release number per megakaryocyte and retained functionality. We show that terminal exposure of in vitro-grown megakaryocytes to the non-specific inhibitor SU6656 significantly increases in vivo yield while leaving in vivo half-life and functionality intact. The exact pathway affected by SU6656 that leads to these results is now being pursued. Disclosures No relevant conflicts of interest to declare.

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Dissecting targeted therapy resistance: Integrating models to quantify environment mediated drug resistance

10.1101/156547 ◽

2017 ◽

Author(s):

Noemi Picco ◽

Erik Sahai ◽

Philip K. Maini ◽

Alexander R. A. Anderson

Keyword(s):

Drug Resistance ◽

Targeted Therapy ◽

Targeted Therapies ◽

Carrying Capacity ◽

Conflicts Of Interest ◽

Treatment Strategies ◽

Model Systems ◽

Significant Heterogeneity

AbstractDrug resistance is the single most important driver of cancer treatment failure for modern targeted therapies. This resistance may be due to the presence of dormant or aggressive tumor cell phenotypes or to context-driven protection. Non-malignant cells and other factors, constituting the microenvironment in which the tumor grows (the stroma), are now thought to play a crucial role in both therapeutic response and resistance. Specifically, the dialogue between the tumor and stroma has been shown to modulate the response to molecularly targeted therapies, through proliferative and survival signaling. The goal of this work is to investigate interactions between a growing tumor and its surrounding stroma in facilitating the emergence of drug resistance. We use mathematical modeling as a theoretical framework to bridge between experimental models and scales, with the aim of separating the intrinsic and extrinsic components of resistance in BRAF mutated melanoma. The model describes tumor-stroma dynamics both with and without treatment. Calibration of our model, through the integration of experimental data, revealed significant variation across animal replicates in either the intensity of stromal promotion or intrinsic tissue carrying capacity. Furthermore our study highlights the need to account for this variation in the design of treatment strategies. Major Findings. Through the integration of a simple mathematical model with in vitro and in vivo experimental growth dynamics of melanoma cell lines (both with and without drug), we were able to dissect the relative contributions of intrinsic versus environmental resistance. Our study revealed significant heterogeneity in vivo, indicating that there is a diversity of either stromal promotion or tumor carrying capacity under targeted therapy. We believe this variation may be one possible explanation for the heterogeneity observed across patients and within individual patients with multiple metastases. Therefore, quantifying this variation both within in vivo model systems and in individual patients could have a significant impact on the design of future treatment strategies that target both the tumor and stroma. Further, we present guidelines for building more effective and longer lasting therapeutic strategies utilizing our experimentally calibrated model. These strategies explicitly consider the protective nature of the stroma and utilize inhibitors that modulate it.PrecisQuantification of the environmental contribution to drug resistance reveals heterogeneity that significantly alters treatment dynamics that can be exploited for therapeutic gain.Financial SupportPicco and Anderson: US National Cancer Institute grant U01CA151924.Picco: UK Engineering and Physical Sciences Research Council (EPSRC grant number EP/G037280/1).Conflict of Interest DisclosureThe authors declare no potential conflicts of interest.

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In vivo CRISPR/Cas9 knockout screen: TCEAL1 silencing enhances docetaxel efficacy in prostate cancer

Life Science Alliance ◽

10.26508/lsa.202000770 ◽

2020 ◽

Vol 3 (12) ◽

pp. e202000770 ◽

Cited By ~ 1

Author(s):

Linda K Rushworth ◽

Victoria Harle ◽

Peter Repiscak ◽

William Clark ◽

Robin Shaw ◽

...

Keyword(s):

Prostate Cancer ◽

Elongation Factor ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Genome Wide ◽

A Genome ◽

Docetaxel Treatment ◽

Crispr Screen ◽

Altered Cell

Docetaxel chemotherapy in metastatic prostate cancer offers only a modest survival benefit because of emerging resistance. To identify candidate therapeutic gene targets, we applied a murine prostate cancer orthograft model that recapitulates clinical invasive prostate cancer in a genome-wide CRISPR/Cas9 screen under docetaxel treatment pressure. We identified 17 candidate genes whose suppression may enhance the efficacy of docetaxel, with transcription elongation factor A–like 1 (Tceal1) as the top candidate. TCEAL1 function is not fully characterised; it may modulate transcription in a promoter dependent fashion. Suppressed TCEAL1 expression in multiple human prostate cancer cell lines enhanced therapeutic response to docetaxel. Based on gene set enrichment analysis from transcriptomic data and flow cytometry, we confirmed that loss of TCEAL1 in combination with docetaxel leads to an altered cell cycle profile compared with docetaxel alone, with increased subG1 cell death and increased polyploidy. Here, we report the first in vivo genome-wide treatment sensitisation CRISPR screen in prostate cancer, and present proof of concept data on TCEAL1 as a candidate for a combinational strategy with the use of docetaxel.

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Leptin Receptor As a Marker for a Subset of Long-Term Functional Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2020-139595 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Thao Trinh ◽

James Ropa ◽

Arafat Aljoufi ◽

Scott Cooper ◽

Edward F. Srour ◽

...

Keyword(s):

Progenitor Cells ◽

Leptin Receptor ◽

Conflicts Of Interest ◽

Gene Set Enrichment Analysis ◽

Type I ◽

Hematopoietic Stem ◽

Therapeutic Implications

The hematopoietic system is maintained by the hematopoetic stem and progenitor cells (HSCs/HPCs), a group of rare cells that reside in a hypoxic bone marrow (BM) microenvironment. Leptin (Lep) is well-known for its neuroendocrine and immunological functions, and its receptor (Lepr) has been studied extensively in the BM niche cells. Yet, its biological implications in HSC/HPC biology remained largely unknown. In this study, we hypothesized that Lepr-expressing HSCs/HPCs are functionally and transcriptomically distinct from their negative counterparts. To test our hypothesis, we utilized both in vitro and in vivo approaches. We first employed Fluorescence-activated cell sorting (FACS) analysis to confirm expression of Lepr on HSCs/HPCs in adult mouse BM. We then isolated equal numbers of Lepr+Lineage-Sca1+cKit+ (LSK cells - a heterogenous population of long-term, short-term HSCs and multipotent HPCs) and Lepr-LSK cells from C57BL/6 (CD45.2+) mouse BM to perform colony-forming unit (CFU) assay and competitive transplantation assay, which also included using competitor cells from BoyJ (CD45.1+) unseparated BM and lethally-irradiated F1 (CD45.1+CD45.2+) as hosts. To determine whether Lepr can further hierarchize HSCs into two distinct populations, we repeated the competitive transplants using freshly isolated C57BL/6 Lepr+HSCs or Lepr-HSCs cells instead. At the end of primary transplants, whole BM were analyzed for donor chimerisms in the peripheral blood (PB) and BM as well as transplanted in a non-competitive fashion into lethally-irradiated secondary recipients. To gain mechanistic insights, we assessed homing potential as homing plays a role in increased engraftment. We also performed bulk RNA-seq using freshly sorted BM Lepr+HSCs or Lepr-HSCs to elucidate potential molecular pathways that are responsible for the differences in their functional capacity. By phenotypic studies, our FACS analyses showed that Lepr+ cells represented a smaller population within the hematopoietic compartment in the BM. However, HSCs contained a higher percentage of Lepr+ cells than other HPC populations. By functional assessments, Lepr+LSK cells were more highly enriched for colony-forming progenitor cells in CFU assay as compared to Lepr-LSK cells. Interestingly, Lepr+LSK cells exhibited more robust engraftment capability in primary transplants and substantial self-renewal capacity in secondary transplants throughout different time points in both PB and BM. In addition, Lepr+HSCs showed significantly higher donor chimerisms in PB month 1, 2, 4 and BM month 4 with similar lineage output compared to Lepr-HSCs. Higher engraftment could be due to increased homing of HSCs to the BM; however, Lepr+HSCs and Lepr-HSCs showed similar homing capacity as well as levels of surface CXCR4 expression. Molecularly, Fast Preranked Gene Set Enrichment Analysis (FGSEA) showed that Lepr+HSCs were enriched for Type-I Interferon and Interferon-gamma response pathways with Normalized Enrichment Scores of 2 or higher. Lepr+HSC transcriptomic study also revealed that these cells as compared to Lepr-HSCs expressed significantly higher levels of genes involved in megakaryopoiesis and proinflammatory immune responses including the NF-κB subunits (Rel and Relb). Interestingly, both IFN-γ and NF-κB signalings have been demonstrated to be critical for the emergence of HSCs from the hemogentic endothelium during embryonic development. In summary, although Lepr+LSK cells occupied a minor fraction compared to their negative counterparts in the BM, they possessed higher colony-forming capacity and were more highly enriched for long-term functional HSCs. In line with this, Lepr+HSCs engrafted significantly higher and self-renewed more extensively than Lepr-HSCs, suggesting that Lepr not only can be used as a marker for functional HSCs but also further differentiate HSCs into two functionally distinguishable populations. Intriguingly, Lepr+HSCs were characterized with a proinflammatory transcriptomic profile that was previously suggested to be critical for the development of HSCs in the embryo. All together, our work demonstrated that Lepr+HSCs represent a subset of highly engrafting adult BM HSCs with an embryonic-like transcriptomic signature. This can have potential therapeutic implications in the field of hematopoietic transplantation as Lepr is highly conserved between mice and human. Disclosures No relevant conflicts of interest to declare.

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RFWD2 Induces Cellular Proliferation and Proteasome Inhibitor Resistance By Mediating p27 Ubiqutinaiton in Multiple Myeloma

Blood ◽

10.1182/blood-2019-127888 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3068-3068

Author(s):

Ye Yang ◽

Mengjie Guo ◽

Chunyan Gu

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Drug Resistance ◽

Proteasome Inhibitor ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Scid Mice ◽

Rpmi 8226

Purpose: In recent years, with the emergence of targeted proteasome inhibitors (PIs), the treatment of multiple myeloma (MM) has made great progress and significantly improves the survival rate of patients. However, MM remains an incurable disease, mainly due to the recurrence of drug resistance. The constitutive photomorphogenic 1 (RFWD2, also known as COP1), is closely related to the occurrence and development of tumors, but its role in MM is largely unknown. This study was aimed to explore the mechanism of RFWD2 on cell proliferation and resistance to proteasome inhibitor in MM. Experimental Design: Using gene expression profiling (GEP) samples, we verified the relation of RFWD2 to MM patients' survival and drug-resistance. The effect of RFWD2 on cell proliferation was confirmed by MTT and cell cycle analysis in RFWD2-overexpressed and RFWD2-knockdown MM cells. MTT and apoptosis experiments were performed to evaluate whether RFWD2 influenced the sensitivity of MM cells to several chemotherapy drugs. MM xenografts were established in immunodeficient NOD/SCID mice by injecting wild-type or RFWD2 over-expression MM cells with drug intervention. The mechanism of drug resistance was elucidated by analyzing the association of RFWD2 with E3 ligase of p27. Bortezomib-resistant RPMI 8226 cells were used to construct RFWD2 knockdown cells, which were injected into NOD/SCID mice to assess the effect of RFWD2 on bortezomib resistance in vivo. Results: RFWD2 expression was closely related to poor outcome, relapse and bortezomib resistance in MM patients' GEP cohorts. Elevated RFWD2 induced cell proliferation, while decreased RFWD2 inhibited cell proliferation and induced apoptosis in MM cells. RFWD2-overexpression MM cells resulted in PIs resistance, however, no chemotherapy resistance to adriamycin and dexamethasone was observed in vitro. In addition, overexpressing RFWD2 in MM cells led to bortezomib resistance rather than adriamycin resistance in myeloma xenograft mouse model. RFWD2 regulated the ubiquitination degradation of P27 by interacting with RCHY1 ubiquitin ligase. The knockdown of RFWD2 in bortezomib-resistant RPMI 8226 cells overcame bortezomib resistance in vivo. Conclusions: Our data demonstrate that elevated RFWD2 induces MM cell proliferation and resistance to PIs, but not to adriamycin and dexamethasone both in vitro and in vivo through mediating the ubiquitination of p27. Collectively, RFWD2 is a novel promising therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

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Investigating Drug Resistance In Leukemia Initiating Cell Populations.

Blood ◽

10.1182/blood.v116.21.1570.1570 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1570-1570

Author(s):

Paraskevi Diamanti ◽

Charlotte Victoria Cox ◽

Allison Blair

Keyword(s):

Oxidative Stress ◽

Drug Resistance ◽

Childhood Leukemia ◽

Conflicts Of Interest ◽

Annexin V ◽

Cell Populations ◽

Childhood All ◽

Nsg Mice ◽

Cd34 Cells

Abstract Abstract 1570 Outcomes for childhood leukemia have improved significantly in recent years with remission rates of over 98% and reported cure rates of 80% for standard risk cases. However, 20% of patients relapse due to failure to eradicate the disease. Further improvement in outcomes will require a better understanding of the biology of this malignancy and the mechanisms of drug resistance. Evidence that several leukemia subpopulations can initiate and maintain this disease in xenograft models and that some of these subpopulations are resistant to current therapeutic agents suggests that relapse may arise from these cells. Parthenolide (PTL), a sesquiterpene lactone compound, has been shown to cause apoptosis in malignant cells by inducing oxidative stress and by inhibiting NF-κB mediated cell survival. In this investigation we have assessed the effects of PTL on leukemia subpopulations in a cohort of childhood ALL cases from mixed prognostic subgroups. Cells from 15 B-ALL cases were stained with antibodies against CD34 and CD19, while CD34 and CD7 were used for 7 T-ALL cases. Cells were then sorted based on expression or lack of expression of the antibody combinations. Unsorted cells and the 4 sorted subpopulations from each type of leukemia were treated with 7.5 and 10 μM PTL for 18–24 hours. The effect of PTL on viability was studied by flow cytometry using Annexin V and Propidium iodide. In B-ALL cases, the CD34+/CD19- population was the least affected with 89.1±6.9% cells surviving PTL treatment. This was significantly higher than the unsorted cells and the other sorted populations (<53%; P<0.01). Most of the T-ALL cases (6/7) were classed as high risk by MRD analyses at day 28. Despite this, unsorted T-ALL cells were more responsive to PTL with only 29.7±12.8% surviving treatment. The CD34+/CD7- population was the least affected (59.9±13.3% viable cells). The functional capacity of the PTL treated unsorted cells and sorted populations was also assessed in vivo. NOD/SCID IL2Rγ null (NSG) mice were inoculated with untreated or PTL treated cells and the levels of engraftment after 10 weeks were compared. The results to date indicate that PTL treatment prevented engraftment of unsorted ALL cells. Mean engraftment levels of 65±20% CD45+ (range 29–99%) were observed using untreated cells while there was no detectable human cell engraftment with the PTL treated cells. This suggests PTL is more effective on unsorted ALL cells than the data from the short term apoptosis assays indicated. Engraftment was achieved using CD34- cells from 3 cases (73±29%, range 40–96%). However, no engraftment was observed when CD34- cells were treated with PTL. In contrast, the levels of engraftment observed with PTL treated CD34+/CD19- B-ALL cells were similar to or greater than those observed with the untreated counterparts (95±8% and 64±9% CD45+ respectively, P≤0.07). The levels of engraftment observed with CD34+/CD7- T-ALL cells were reduced with PTL treatment from 67±21% to 12±9% CD45+ (P≤0.03) but not eliminated. Subsequently, we investigated the mechanisms for this apparent resistance to PTL in the primitive cell populations. PTL has been associated with induction of oxidative stress, activation of p53 and inhibition of NF-κB in AML and CLL. We used confocal microscopy to investigate whether NF-κB is constitutively expressed in ALL cases and to evaluate the effect of PTL on the phosphorylation of NF-κB. Three B-ALL and 3 T-ALL cases, where the unsorted populations had been affected by PTL while the respective CD34+/CD19- and CD34+/CD7- populations were more resistant, were investigated. Cells were stained with anti-phospho-p65 polyclonal antibody and Alexa fluor 488. NF-κB was constitutively activated in all cases. There was evidence of decreased phosphorylation in unsorted PTL treated cells indicating inhibition of NF-κB. However, in the phenotypically primitive cells there was no difference in the phosphorylation levels compared to untreated cells or phosphorylation was increased. This suggests NF-κB was not inhibited, which could explain the observed resistance of these leukemia populations to PTL. These data demonstrate that some leukemia initiating cell populations in childhood B-ALL and T-ALL are resistant to PTL. A more thorough understanding of these leukemia initiating cell populations and their mechanisms of resistance will be required for the development of more effective therapies. Disclosures: No relevant conflicts of interest to declare.

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Assessing CD97 and CD99 As Markers of Leukaemia Initiating Cells in Paediatric ALL

Blood ◽

10.1182/blood.v120.21.1882.1882 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1882-1882 ◽

Cited By ~ 1

Author(s):

Charlotte Victoria Cox ◽

Paraskevi Diamanti ◽

Allison Blair

Keyword(s):

Functional Capacity ◽

Conflicts Of Interest ◽

Residual Disease ◽

Survival Rates ◽

In Vivo Studies ◽

Nsg Mice ◽

Early Treatment Response ◽

Current Therapies

Abstract Abstract 1882 Overall survival rates in paediatric acute lymphoblastic leukaemia (ALL) have dramatically improved but around 20% do not respond to current therapies and subsequently relapse. Leukaemia initiating cells (LIC) are the topic of much investigation, as these cells can self-renew and may have the potential to cause relapse. It has been shown that multiple subpopulations of ALL cells have the ability to initiate the disease in immune deficient mouse models. Therefore, treatment should be targeted at all cells with this capacity, if the disease is to be eradicated. Minimal residual disease (MRD) detection is an invaluable tracking tool to assess early treatment response and recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. CD97 and CD99 are two markers which were over expressed in paediatric ALL. Incorporating these markers into investigations of LIC may allow discrimination of leukaemia cells from normal haemopoietic stem cells (HSC). In this study we evaluated the expression of CD34 in combination with CD97 in B cell precursor (BCP) ALL cases and CD99 in T-ALL cases and subsequently assessed the functional capacity of the sorted subpopulations in vitro and in vivo. Ten ALL samples (6 B-ALL & 4 T-ALL) with a median age 7 years (range 2–15 years) were studied. One B-ALL case and 3 T-ALL cases were considered high risk by molecular assessment of MRD at day 28 of treatment. Flow cytometric analyses of the ALL samples and 8 normal haemopoietic cell samples demonstrated that both CD97 and CD99 were over expressed in ALL patients (78.9±14.8% & 76.4±32.8%, respectively) when compared to normal haemopoietic cells (14.1±25.4%; p=0.001, 47.1±10%; p=0.03, respectively). Cells were sorted for expression/lack of expression of these markers and proliferation of the sorted cells was assessed in suspension culture over a 6 week period. In the B-ALL patients the CD34+/CD97+ subpopulation represented the bulk of leukaemia cells (65.2±32.1%), the CD34−/CD97+ the smallest fraction (3.3±2.4%) with the CD34+/CD97− and CD34−/CD97− subpopulations representing 21.1±31.5% and 10.5±5.8% of cells, respectively. When the functional capacity of these subpopulations was assessed in vitro greatest expansion was observed in cells derived from CD34+/CD97− subpopulation (2–173 fold) from 9.4×103 at initiation up to 1.5×106 cells at week 6. Expansion was also observed, to a lesser extent in the CD34−/CD97− subpopulation (3.4–28 fold) from 8×103 up to 1.4×106 cells. No expansion was observed in cultures of CD34+/CD97+ and CD34−/CD97− subpopulations but cells were maintained throughout the culture period. These sorted subpopulations were also inoculated into NOD/LtSz-SCID IL-2Rγc null (NSG) mice to evaluate repopulating capacity. To date, engraftment has been achieved with 3 subpopulations; CD34+/CD97+ (3–28.8% CD45+), CD34+/CD97− (0.5–25.5% CD45+) and CD34−/CD97+ (23.8% CD45+) cells. When the functional capacity of T-ALL cases was assessed the CD34+/CD99+ subpopulation represented the bulk of cells at sorting (51.87±47.2%), the CD34+/CD99- subpopulation was the smallest (0.9±0.8%) and the CD34−/CD99+ and CD34−/CD99− subpopulations represented 32.1±38.9% and 27.2±33.4% of cells, respectively. Greatest expansion was observed in cultures of CD34+/CD99- cells (4.6–1798 fold) from 7.5×103 up to 2.6×106 cells at week 6. The other 3 subpopulations expanded to a lesser extent (1.3–216 fold) from 5×103 up to 1.8×106 cells. When the functional capacity of these cells was assessed in NSG mice, engraftment was achieved in all subpopulations; CD34+/CD99+ (87–90.5% CD45+), CD34+/CD99− (1.5–84.9% CD45+), CD34−/CD99+ (31.3–98.6% CD45+) and CD34−/CD99− (3–92.9% CD45+). In some cases, cells recovered from BM of NSG inoculated with CD99− cells had high expression of CD99, typical of the patient samples at diagnosis, indicating that the inoculated CD99− cells had differentiated in vivo. Studies are ongoing to assess the self-renewal capacity of these subpopulations by serial transplantation. The findings to date indicate that targeting CD97 and CD99, either alone or in combination with CD34 would not eliminate all cells with the capacity to initiate and maintain B-ALL and T-ALL, respectively. Further developments in therapy may require targeting leukaemogenic pathways, rather than only cell surface markers to improve survival outcome in paediatric ALL. Disclosures: No relevant conflicts of interest to declare.

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FOXM1, CDK6 and Rb Dependent Drug Resistance and Senescence in Myeloma

Blood ◽

10.1182/blood.v128.22.4456.4456 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4456-4456 ◽

Cited By ~ 1

Author(s):

Chunyan Gu ◽

Ruini Chen ◽

Xuefang Jing ◽

Siegfried Janz ◽

Ye Yang

Keyword(s):

Drug Resistance ◽

High Risk ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Physical Interaction ◽

Mrna Levels ◽

Agar Culture ◽

Myeloma Cells

Abstract We recently reported that FOXM1 is a promising therapeutic target in multiple myeloma (MM), particularly for the subset of patients with high-risk disease.Because high-risk myeloma exhibits a strong predilection to early drug-resistant relapse following first-line therapy, we here decided to evaluate the role of FOXM1 in the acquisition of drug resistance by myeloma cells. We analyzed gene expression profiles of 88 paired myeloma samples at baseline and relapse from the UAMS Total Therapy 2 cohort and found that FOXM1 mRNA levels were significant upregulated in relapsed myeloma and that this was associated with poor event-free and overall survival. Laboratory studies showed that enforced expression of FOXM1 in human myeloma cell lines (HMCLs) results in decreased sensitivity of cells to widely used myeloma drugs, such as bortezomib and doxorubicin. This was observed in vitro, in both bulk cell and soft-agar culture, and in vivo using xenografting in mice. Biochemical analysis of HMCLs revealed physical interaction of FOXM1 with CDK6 and Rb, key regulators of cell cycle progression and cellular senescence, respectively.Treatment with small-compound CDK6 inhibitor, inhibited myeloma growth, decreased clonogenicity of myeloma, and ameliorated FOXM1-dependent senescence. Genetic and pharmacological targeting of FOXM1 in myeloma cells using shRNA and thiostreptone respectively, led to growth arrest and senescence, while elevated expression of FOXM1 reversed these phenotypes. In sum, our findings implicating the FOXM1-CDK6-Rb network in drug resistance and senescence of high-risk myeloma point to new treatment opportunities for this difficult-to-cure neoplasm. Disclosures No relevant conflicts of interest to declare.

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Genome-wide CRISPR Screen to Identify Genes that Suppress Transformation in the Presence of Endogenous KrasG12D

Scientific Reports ◽

10.1038/s41598-019-53572-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Jianguo Huang ◽

Mark Chen ◽

Eric S. Xu ◽

Lixia Luo ◽

Yan Ma ◽

...

Keyword(s):

Kinase Inhibitor ◽

Gene Mutations ◽

Soft Agar ◽

Reading Frame ◽

Immunodeficient Mice ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

AbstractCooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.

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Whole-Genome CRISPR Screen Reveals the Mechanism of Relapse in Patient-Derived Cells Representing High-Risk Paediatric ALL

Blood ◽

10.1182/blood-2019-124818 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3952-3952

Author(s):

Katarzyna Szoltysek ◽

Helen Blair ◽

Sirintra Nakjang ◽

Ricky Tirtakusuma ◽

Mankaran Singh ◽

...

Keyword(s):

High Risk ◽

Research Funding ◽

Ex Vivo ◽

Mtor Inhibitors ◽

Whole Genome ◽

Nsg Mice ◽

Genome Wide ◽

Crispr Screen ◽

Dexamethasone Resistance

Acute lymphoblastic leukemia (ALL) is the most common type of childhood leukaemia. Recently improved risk stratification resulted in therapy optimization and extended survival for the majority of cases. Unfortunately, there is still a significant number of patients either relapsing or not responding to treatment with response to glucocorticoids being one of the most important prognostic indicators of treatment outcome. In order to investigate the mechanism of dexamethasone resistance, we performed genome-wide CRISPR screens in patient derived xenotransplant (PDX) material from t(17;19)-positive ALL. Primary material was obtained from the patient at the presentation and at relapse stage of disease and corresponding PDX samples were generated in immunocompromised NSG mice. PDX cells were lentivirally transduced with the CRISPR knockout pooled 'Brunello' library and then subjected to dexamethasone pressure both ex vivo and in vivo. For the in vivo screen, CRISPR-modified cells were intrafemorally injected into immunodeficient NSG mice followed by either 7.5mg/kg dexamethasone or vehicle treatment. In parallel, PDXs were co-cultured with mesenchymal and endothelial-like human stromal cells generated from human bone marrow-derived iPSCs. Data analysis performed with the MAGeCKFlute software identified the glucocorticoid receptor gene NR3C1 as a main driver of chemoresistance-mediated relapse in this high-risk ALL. Notably, a homozygous deletion of NR3C1 was present in the relapse PDX sample. Furthermore, we identified that loss of the NR3C1 gene in those cells was associated with an inferior engraftment potential in the absence of dexamethasone. Interestingly, the whole-genome CRISPR screen in the relapse sample identified BCL2 and several genes associated with the mTOR pathway as crucial for leukaemic propagation. Knockout of NR3C1 in the diagnostic PDX also established dexamethasone resistance and further enhanced the already significant sensitivity towards mTOR inhibitors. To explore a potential synergism between BCL2 and mTOR inhibition, we assessed the effect of the BCL2 inhibitor ABT-199 and several mTOR inhibitors in both presentation and relapse PDX samples. PDX samples were co-cultured with MSCs and treated with drug combinations in a matrix format for 96 hrs followed by high-throughput fluorescence microscopy-based analysis. These experiments revealed substantial synergism of ABT-199 and mTOR inhibitors associated with increased cell death and prolonged growth inhibition in both presentation and relapse samples. In conclusion, our studies (i) demonstrate that genome-wide CRISPR screens are feasible in PDX material both ex vivo and in vivo, (ii) provide an explanation for the relative rarity of NR3C1 mutations in relapsed material and (iii) identify drug combinations effective in both diagnostic and relapse PDX for further preclinical evaluation. Disclosures Vormoor: Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.

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