Mutant TP53 prevents Telomere Shortening in Acute Myeloid Leukemia

Abstract Background High telomerase activity represents a critical feature of hematopoietic stem cells. Excessive shortening of telomere length (TL) due to replicative stress may be - in analogy to many solid tumors - a hallmark of myeloid neoplasia (MN). Also, telomeric footprints in leukemic genomes may vary between various subtypes corresponding to the differentiation arrest at various stages of hematopoietic ontogeny or specific molecular defects. Critical TL shortening has been associated with genomic instability and accelerated acquisition of genomic lesions leading to a more aggressive phenotype. These processes have not been systematically studied in MN, especially AML. Aim By taking advantage of next-generation sequencing to assay both molecular features and TL within large cohorts of patients, we tested the hypothesis that TL shortening is excessive in highly proliferative MN, but that distinct invariant differences characterize genetic subtypes. Methods Our cohort included AML (N=734), MDS (N=701), healthy controls (HC) (N=11) and PNH (N=102) serving as clonal non-malignant controls. All patients were diagnosed according to WHO standards before being subjected to transcriptome (WTS) and genome (WGS, 100x) sequencing. To retrieve TL characteristics and telomere repeat heterogeneity from WGS data, we used TelomereHunter (TH). In parallel, we performed C-Circle assays. Patients were annotated for clinical features and analyzed for genetic/transcriptomic patterns. Results For a subset of patients for whom corresponding benign lymphocyte DNA was available a significant TL shortening in blasts vs control lymphocytes (A; P=.0023) was detected. While age correlation was established in controls, despite a trend, in MN age did not significantly affect TL (B) and thus subsequent comparisons were not adjusted for age. Next, we studied a cohort of patients with AML, MDS, PNH and HC and found that TL shortening was an overarching finding in AML, MDS and PNH as compared to HC (C). Since no matched DNA was available as reference, we examined the distribution of TL across different age cohorts, AML patients divided according to age cohorts harbored TL in a similar range (D, P=.057). Classic morphologic (E) or cytogenetic subtypes AML exhibited no difference. Similarly, no differences were found between high and low risk MDS patients (not shown). The variability of TL ranges suggested that there may be molecular factors which affect individual TL. When we compared TL grouped according to frequent mutations, only TP53 mutations were associated with longer TL (F, P<.0001). A significant positive correlation (G, P=.021) between TL and TP53 clonal burden was found; samples with the longest vs shortest TL showed significantly higher TP53 VAF (H, P=.0229). In analogy, the presence of multiple TP53 mutations (putative biallelic inactivation) showed longer TL than single hits but no association was found between the nature of mutations and TL (I, J, K). Availability of WTS data allowed us to assess the telomerase activity using the EXTEND score (ES) which has been shown to assess telomerase activity. Indeed, the ES was correlated with TL (L) and TP53 mutant status was associated with a higher ES compared to WT samples (M, P<.0001). Similarly, because of the compensatory upregulation of TP53 in mutant cases, we have also found that TP53 mRNA levels correlated with ES (N P<.0001). Another explanation of TL increase could be the occurrence of alternative lengthening (ALT). TH software allows for estimation of the abundance of specific telomeric repeats. Singleton analysis showed that increase in telomere repeats variants (TTTGGG, O, P=.003) was related to mutations in TP53 arguing against the involvement of ALT. The final confirmation that TL extension was not due to ALT was provided by C-Circle assays. When C-Circle assays were performed for samples with a high/low TL and mutant/WT TP53, none of the subgroups was identified as ALT + (P). Conclusion We stipulate that TL measurements using NGS will be helpful to investigate pathophysiological features associated with TL shortening. Availability of therapies targeting the telomere machinery (Imetelstat) may offer an opportunity for personalized therapy beyond MPN, its current indication. It remains to be tested whether long TL associated with TP53 mutations can serve as marker of sensitivity or resistance to these agents. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy; Novartis: Consultancy.

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Aberrant Telomere Length and Composition Are Recurrent Features of Myeloid Disorders

Blood ◽

10.1182/blood-2020-140662 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-30

Author(s):

Adam Wahida ◽

Stephan Hutter ◽

Manja Meggendorfer ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Telomere Length ◽

Healthy Subjects ◽

Telomerase Activity ◽

Telomere Maintenance ◽

Outcome Analysis ◽

Critical Threshold ◽

Common Mutation ◽

Altered Expression ◽

Telomere Repeat ◽

Tp53 Mutations

Background: In cancer evolution, development of telomere maintenance mechanisms represents a critical feature to achieve replicative immortality and prevent cell death after shortening below a critical threshold. Several studies have indicated that excessive telomere shortening or aberrant telomeric repeats are present in cancer genomes, however, their precise role in myeloid malignancies remains largely unexplored. We stipulated that immature leukemia-initiating cells (LIC) would display higher telomerase activity and thus longer telomeres while in mature LIC lower telomerase activity would be associated with shorter telomeres due to excessive division rate. Additional lesions may also lead to disturbed differentiation programs resulting in altered expression of telomerase. Taking advantage of a vast cohort (N=1648) of patients with myeloid neoplasia analyzed by whole genome sequencing (WGS, mean. coverage 100x) we determined telomere length (TL). Aim: We set out to hypothesize that WGS data can be used to reliably measure the TL and that if systematically applied to well defined patient cohorts, we could test whether preservation of TL or its excessive shortening are prognostically or biologically relevant to clinical outcome. Methods: Our cohort included patients with AML (N=732), MDS (N=701) and MPN (N=102 incl. CML, ET, PMF, PV). Healthy subjects (N=11) and patients with the clonal non-malignant HSC disorder PNH (N=102) served as controls. To retrieve TL and telomere repeat heterogeneity via singleton analysis from WGS data we used two independent bioinformatic tools TelomereHunter and Telseq (1A). Results: First, using bidirectional cross-confirmation with independent bioinformatics pipelines, reliability of WGS-based TL measurements (1B) was confirmed and, as anticipated, age-dependency of TL in healthy controls was reproduced (1C). Next we investigated age as a possible confounder of TL shortening in patients. While in PNH age had an expected impact, patients with AML or MDS exhibited TL decoupled from its inherent age-dependence (r=.00034, P=.72). In general, we noted TL shortening as an overarching feature of myeloid proliferation irrespective of entity (1D). Furthermore, mutations in the promotor region of TERT were not correlated with TL. In AML or MDS, neither morphological nor genetic subclassification where associated with differences in TL (1E), with the exception of a small subset of AML and MDS patients (n=103, n=97), harboring abnormally long telomeres compared to healthy subjects. These were significantly associated with mutations in TP53 (AML P=1.91x10-6, mean TL 558.4; MDS P=.55x10-7, mean TL 414.5) but not with any other common mutation. This is surprising, since TP53 mutations are thought to protect cells from telomeric catastrophe following excessive shortening. Moreover, comparing the VAF of TP53mut AML samples with the corresponding measured TL, revealed a positive correlation (r=.2281, P=.021), irrespective of the blast count by cytomorphology (1F). Singleton analysis from all 1648 patients revealed that in AML and MDS patients, alternative telomere repeat- variants (chiefly TTTGGG) were increased pointing towards a possible implication of alternative telomere extension (1G). Lastly, outcome analysis revealed that in AML, the presence of longer telomeres (compared to controls) was associated with worse prognosis (P=.0035) (1H). Multivariate analysis, which included age, karyotype, TP53 status and gender, revealed that this was due to the strong association with TP53 mutations (n=630 vs n=103). The implications of our study extend beyond the realm of telomere biology and leukemia pathophysiology. Therapies targeting the telomere machinery, e.g. imetelstat, may offer an opportunity for application beyond MPN, its current indication. Ongoing studies are currently investigating its use in MDS. Based on our findings we argue that treatment may be rationally evaluated in TP53mut AML. Conclusion: Our findings provide a rationale for (1) the use of WGS to investigate TL, (2) the fundamental discovery that TL shortening is a common principle in myeloid neoplasms, (3) TP53 mutations being accompanied with increased TL and (4) the exploitation of TL to identify subgroups eligible for treatment with imetelstat. Moreover, WGS-based TL analysis in leukemias can contribute to our understanding of TL maintenance mechanisms in leukemic genome. Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.

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Delineating spatiotemporal and hierarchical development of human fetal innate lymphoid cells

Cell Research ◽

10.1038/s41422-021-00529-2 ◽

2021 ◽

Author(s):

Chen Liu ◽

Yandong Gong ◽

Han Zhang ◽

Hua Yang ◽

Yang Zeng ◽

...

Keyword(s):

Single Cell ◽

Progenitor Cells ◽

Fetal Liver ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Lymphoid Tissues ◽

Hematopoietic Stem ◽

Molecular Features ◽

Lymphoid Progenitors ◽

B Lineage

AbstractWhereas the critical roles of innate lymphoid cells (ILCs) in adult are increasingly appreciated, their developmental hierarchy in early human fetus remains largely elusive. In this study, we sorted human hematopoietic stem/progenitor cells, lymphoid progenitors, putative ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 post-conception weeks, for single-cell RNA-sequencing, followed by computational analysis and functional validation at bulk and single-cell levels. We delineated the early phase of ILC lineage commitment from hematopoietic stem/progenitor cells, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor as a surface marker for the lymphoid progenitors in fetal liver with T, B, ILC and myeloid potentials, while IL-3RA– lymphoid progenitors were predominantly B-lineage committed. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the proliferating characteristics shared by the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2– CCR9+ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.

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Accelerated telomere shortening in glycosylphosphatidylinositol (GPI)–negative compared with GPI-positive granulocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) detected by proaerolysin flow-FISH

Blood ◽

10.1182/blood-2004-10-3996 ◽

2005 ◽

Vol 106 (2) ◽

pp. 531-533 ◽

Cited By ~ 29

Author(s):

Fabian Beier ◽

Stefan Balabanov ◽

Tom Buckley ◽

Klaus Dietz ◽

Ulrike Hartmann ◽

...

Keyword(s):

Telomere Length ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Failure ◽

Disease Stage ◽

Healthy Individuals ◽

Hematopoietic Stem ◽

Replicative Stress ◽

Selective Growth Advantage ◽

Bone Marrow Failure Syndromes ◽

The Impact

Abstract Telomere length has been linked to disease stage and degree of (pan-)cytopenia in patients with bone marrow failure syndromes. The aim of the current study was to analyze the impact of replicative stress on telomere length in residual glycosylphosphatidylinositol-positive (GPI+) versus GPI– hematopoiesis in patients with paroxysmal nocturnal hemoglobinuria (PNH). Peripheral blood granulocytes from 16 patients and 22 healthy individuals were analyzed. For this purpose, we developed proaerolysin flow-FISH, a novel methodology that combines proaerolysin staining (for GPI expression) with flow-FISH (for telomere length measurement). We found significantly shortened telomeres in GPI– granulocytes (mean ± SE: 6.26 ± 0.27 telomere fluorescence units [TFU]), both compared with their GPI+ counterparts (6.88 ± 0.38 TFU; P = .03) as well as with age-matched healthy individuals (7.73 ± 0.23 TFU; P < .001). Our findings are in support of a selective growth advantage model of PNH assuming that damage to the GPI+ hematopoietic stem-cell (HSC) compartment leads to compensatory hyperproliferation of residual GPI–HSCs.

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Myc and Human Papillomavirus Type 16 E7 Genes Cooperate To Immortalize Human Keratinocytes

Journal of Virology ◽

10.1128/jvi.00669-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12689-12695 ◽

Cited By ~ 27

Author(s):

Xuefeng Liu ◽

Gary L. Disbrow ◽

Hang Yuan ◽

Vjekoslav Tomaić ◽

Richard Schlegel

Keyword(s):

Transcriptional Activation ◽

High Efficiency ◽

P53 Protein ◽

Significant Proportion ◽

Telomerase Activity ◽

Human Papillomaviruses ◽

Mrna Levels ◽

Crisis Period ◽

Cell Immortalization ◽

Immortalized Cells

ABSTRACT The E6 protein of the oncogenic human papillomaviruses (HPVs), in combination with the E7 protein, is essential for the efficient immortalization of human foreskin keratinocytes (HFKs). Since we recently demonstrated that E6 activates the human telomerase reverse transcriptase (hTERT) promoter via a Myc-dependent mechanism, we speculated that overexpressed Myc might be able to substitute for E6 in cell immortalization. Myc (similar to E6) was unable to immortalize HFKs when transduced alone, despite inducing high levels of telomerase activity. However, when transduced with E7, Myc immortalized HFKs following a brief but detectable crisis period. In contrast to E6 + E7-immortalized cells, the Myc + E7-immortalized cells expressed high levels of p53 protein as well as two p53-regulated proteins, p21 and hdm-2. The increase in p21 and hdm-2 proteins correlated directly with their mRNA levels, suggesting transcriptional activation of the respective genes by the overexpressed p53 protein. Interestingly, a significant proportion of the p53 protein in the Myc + E7-immortalized cells was localized to the cytoplasm, potentially due to interactions with the overexpressed hdm-2 protein. Regardless, cell immortalization by the Myc + E7 genes occurred independently of p53 degradation. Since we have already observed high-efficiency cell immortalization with the hTERT + E7 or E6 mutant (p53 degradation-defective) + E7 genes (i.e., no crisis period) that proceeds in the presence of high levels of p53, we hypothesize that the crisis period in the Myc + E7 cells is due not to the levels of the p53 protein but rather to unique properties of the Myc protein. The common factor in cell immortalization by the three gene sets (E6 + E7, Myc + E7, and hTERT + E7 genes) is the induction of telomerase activity.

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B-Cell Acute Lymphoblastic Leukemia Arising in Patients with a Preexisting Diagnosis of Multiple Myeloma Is a Novel Cancer with High Incidence of TP53 Mutations

Blood ◽

10.1182/blood-2020-142067 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 20-20

Author(s):

Monique Chavez ◽

Erica Barnell ◽

Malachi Griffith ◽

Zachary Skidmore ◽

Obi Griffith ◽

...

Keyword(s):

Multiple Myeloma ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Mechanism Of Action ◽

Lymphoblastic Leukemia ◽

Treatment Options ◽

Hematopoietic Stem ◽

Tp53 Mutations ◽

Cell Acute Lymphoblastic Leukemia

Multiple Myeloma (MM) is a malignancy of plasma cells that affects over 30,000 Americans every year. Despite advances in the treatment of the disease, approximately 12,000 American patients will still die of MM in 2019. One of the mainstays of treatment for MM is the immunomodulatory and antiangiogenic drug lenalidomide; which is used in induction therapy, maintenance therapy and treatment of relapsed disease. Although not fully elucidated, lenalidomide's mechanism of action in MM involves the drug binding to Cerebelon (CBN) and leads to the subsequent degradation of the Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors (TF). These TFs play important regulatory roles in lymphocyte development. Despite lenalidomide's importance in MM treatment, several groups have reported that MM patients treated with lenalidomide rarely go on to develop B-cell acute lymphoblastic leukemia (B-ALL). The genetics and clonal relationship between the MM and subsequent B-ALL have not been previously defined. Importantly, it is not clear if the MM and B-ALL arise from the same founding clone that has been under selective pressure during lenalidomide treatment. As deletions in IKZF1 are common in B-ALL, one could hypothesize that lenalidomide's mechanism of action mimics this alteration and contributes to leukemogenesis. We sequenced the tumors from a cohort of seven patients with MM treated with lenalidomide who later developed B-ALL. These data did not show any mutational overlap between the MM and ALL samples-the tumors arose from different founding clones in each case. However, several genes were recurrently mutated in the B-ALL samples across the seven patients. These genes included TP53, ZFP36L2, KIR3DL2, RNASE-L, and TERT. Strikingly, five of the seven patients had a TP53 mutations in the B-ALL sample that was not present in the matched MM sample. The frequency of TP53 mutations in our cohort was much higher than that reported in adult de novo B-ALL patients which can range between 4.1-6.4% (Hernández-Rivas et al. 2017 and Foa et al. 2013). Utilizing CRISPR-Cas9 gene editing, we disrupted the Zfp36l2 or Actb in murine hematopoietic stem cells (HSCs) of mice with or without loss of Trp53. We performed our first transplantation experiment in which the cohorts of mice have loss of Trp53 alone, loss of Zfp36l2 alone, loss of both Trp53 and Zfp36l2, or a control knockout (KO) of Actb. To characterize the disruption of Zfp36l2 alone and in combination with Trp53 we analyzed the hematopoietic stem and progenitor cell compartments in the bone marrow of the above transplanted mice. In mice with a loss of Zfp36l2 there is a decrease in Lin- Sca-1+ c-Kit+ (LSK), short term-HSC (ST-HSC), and multipotent progenitors (MPP). This decrease was not observed in the mice with a loss of both Trp53 and Zfp36l2, where instead we noted an increase in monocyte progenitors (MP), granulocytes-macrophage progenitors (GMP), and common myeloid progenitors (CMP) cells. In this Trp53 Zfp36l2 double loss model we also noted a decrease in B220+ B-cells that was not seen in the Zfp36l2 alone. In this cohort of Trp53 Zfp36l2 loss, we characterized B-cell development through hardy fraction flow cytometry, and identified a decrease in fractions A and B/C (pre-pro and pro-B-cells, respectively) as compared to Zfp36l2 or Actb alone. As lenalidomide does not bind to Cbn in mice, we used the human B-ALL NALM6 cell line to test if treatment with lenalidomide will lead to a selective growth advantage of cells with the same genes knocked out versus wild-type control cells grown in the same culture. We hypothesize that lenalidomide treatment selectively enriched for pre-existing mutated cell clones that evolved into the B-ALL. Preliminary data in NALM6 cells with a loss of TP53 demonstrate a slight increase in cell number at day 7 compared to a RELA control. These experiments will be repeated with concurrent ZFP36L2 and TP53 mutations as well as ZFP36L2 alone. Treatment-related disease is a key consideration when deciding between different treatment options, and this project aims to understand the relationship between MM treatment and B-ALL occurrence. It may be possible to identify MM patients who are at-risk for B-ALL. For example, MM patients who harbor low-level TP53 mutations prior to lenalidomide treatment could be offered alternative treatment options. Disclosures Barnell: Geneoscopy Inc: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Wartman:Novartis: Consultancy; Incyte: Consultancy.

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Relationship between Protein kinase C isoforms, Telomerase and Alpha- fetoprotein through PI3K/AKT/mTOR pathway in Hepatocellular carcinoma

MedPharmRes ◽

10.32895/ump.mpr.5.4.3 ◽

2021 ◽

Vol 5 (4) ◽

pp. 12-26

Author(s):

Rita Ammoury ◽

Roula Tahtouh ◽

Nadine Mahfouz ◽

Raia Doumit ◽

Charbel Khalil ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Protein Kinase C ◽

Protein Kinase ◽

Telomerase Activity ◽

Mtor Pathway ◽

Alpha Fetoprotein ◽

Mrna Levels ◽

Htert Expression ◽

Kinase C ◽

Pkc Isoforms

Protein kinase C (PKC) family has been an alluring objective for new cancer drug discovery. It has been reported to regulate telomerase in several cancer types. Our team had previously used telomerase to elucidate alpha-fetoprotein (AFP) modulation in hepatocellular carcinoma (HCC). The aim of this study was to investigate the interrelationships among PKC isoforms, telomerase and AFP in HCC. PKCα and PKCδ were the most expressed isoforms in HepG2/C3A, PLC/PRF/5, SNU-387 and SKOV-3 cells. Following the upregulation of AFP using pCMV3-AFP and the human telomerase reverse transcriptase (hTERT) using a construct expressing a wild-type hTERT, and after their inhibition with all-trans retinoic acid and hTERT siRNA each respectively, we found that the expression of PKCα, PKCβI, PKCβII and PKCδ was affected by the variation of AFP and hTERT mRNA levels. An increase in AFP expression and secretion was observed after gene silencing of PKCα, PKCβ, PKCδ, and PKCε in HepG2/C3A. A similar pattern was observed in transfected PLC/PRF/5 cells, however PKCδ isoform silencing decreased AFP expression. Furthermore, telomerase activity was quantified using quantitative telomeric repeat amplification protocol. The variations in hTERT expression and telomerase activity were similar to those of AFP. Further investigation showed that PKC isoforms regulate AFP and hTERT expression levels through PI3K/AKT/mTOR pathway in HepG2/C3A and PLC/PRF/5 cells. Thus, these results show for the first time a possible interrelationship that links PKC isoforms to both AFP and hTERT via PI3K/AKT/mTOR pathway in HCC.

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Dynamic Telomere Shortening and Chromosomal Instability in Irradiated CD34+ Cells Transduced with TP53 Hotspot Mutations R175H, R248W and R249S

Blood ◽

10.1182/blood.v126.23.4832.4832 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4832-4832

Author(s):

Azam Salari ◽

Kathrin Thomay ◽

Andrea Schienke ◽

Maike Hagedorn ◽

Juliane Ebersold ◽

...

Keyword(s):

Chromosomal Instability ◽

Tp53 Mutation ◽

Lower Amount ◽

Complex Karyotype ◽

Telomere Shortening ◽

Hematopoietic Stem ◽

Tp53 Mutations ◽

Ht1080 Cells ◽

Stem And Progenitor Cells ◽

Hotspot Mutations

Abstract Patients with MDS and a complex karyotype have a very short median survival and a high risk of transformation into AML. We showed earlier that TP53 mutations are associated with complex karyotype and disease progression. However, it is poorly understood how TP53 mutations contribute to the induction of chromosomal instability in hematopoietic stem and progenitor cells. We therefore established a long-term cell culture (LTC) model and investigated the role of TP53 in human CD34+ hematopoietic stem and progenitor cells (HSCs) isolated from cord blood and in HT1080 cells over 6 weeks. We chose 3 different modifications: 1) TP53-deficient HSCs via shRNA knockdown, 2) HSCs with different lentivirally introduced TP53 hotspot mutations (R248W, R175H, R273H, R249S) and 3) HT1080 cells with different lentivirally introduced TP53 hotspot mutations (R248W, R175H, R273H, R249S). We performed each LTC at least three times. In order to stress the cells and induce chromosomal instability, we irradiated half of the cells. Besides functional assays in the first week, we performed detailed cytogenetic analysis including telomere length measurement at weeks 1, 3 and 6. TP53 mutations and the downregulation of TP53 led to impaired hematopoiesis with decreased erythroid differentiation, increased apoptosis and decreased proliferation. None of the modifications induced chromosomal instability in cells without irradiation. In the irradiated cells, all cells carrying a TP53 mutation or a TP53 downregulation developed a chromosomal instability in comparison to the cells transduced with control vectors. However, no stable complex clones developed. Telomeres shortened during follow-up in HSCs carrying the mutations R175H, R248W and R249S. No other cells showed a dynamic response in telomere length. In order to analyze the DNA repair capacity, we performed yH2AX foci assay. Surprisingly, the same cells which showed a telomere shortening showed a lower amount of foci. This could be due to a lower amount of double-strand breaks or to a lower ability to form foci. In summary, TP53 mutations and the downregulation of TP53 led to an increased chromosomal instability in irradiated cells only. Modifications alone did not lead to the development of complex karyotypes. Furthermore, only the mutations R175H, R248W and R249S led to a telomere shortening. In conclusion, a TP53 mutation seems to require additional passenger mutations in order to lead to MDS or AML with complex karyotype. Disclosures No relevant conflicts of interest to declare.

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TP53 Action and the Consequences of TP53 Mutation in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-121285 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. SCI-13-SCI-13

Author(s):

Scott W. Lowe

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Tp53 Mutation ◽

P53 Mutation ◽

Tp53 Mutations ◽

Genomic Analyses ◽

Equity Ownership ◽

Myeloid Neoplasia ◽

The Impact ◽

Acute Myeloid

p53 action and the consequences of p53 mutation in acute myeloid leukemia TP53 mutations are common in treatment associated myeloid neoplasia (tMN) and complex karyotype acute myeloid leukemia (CK-AML), where they are associated with chemoresistance and one of the worst prognoses of any leukemia genotype. To understand the impact of TP53 mutations on AML biology, we are performing arge scale genomic analyses of p53 mutant AML and have produced a series of animal models that appear to faithfully reflect molecular and biological features of the human disease. We have gone on to explore the biology of particular TP53 mutational configurations drive AML initiation and maintenance, and to identify and understanding the events that cooperate with p53 mutations during leukemogenesis. Disclosures Lowe: Blueprint Medicines: Consultancy, Equity Ownership; ORIC pharmaceuticals: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership.

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Thrombopoietin Induces HOXA9 Nuclear Transport in Immature Hematopoietic Cells: Potential Mechanism by Which the Hormone Favorably Affects Hematopoietic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.24.15.6751-6762.2004 ◽

2004 ◽

Vol 24 (15) ◽

pp. 6751-6762 ◽

Cited By ~ 53

Author(s):

Keita Kirito ◽

Norma Fox ◽

Kenneth Kaushansky

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Hematopoietic Stem Cells ◽

Nuclear Translocation ◽

Hematopoietic Cells ◽

Mitogen Activated Protein Kinase ◽

Mrna Levels ◽

Hematopoietic Stem ◽

Nuclear Localization Signals ◽

Mitogen Activated Protein

ABSTRACT Members of the homeobox family of transcription factors are major regulators of hematopoiesis. Overexpression of either HOXB4 or HOXA9 in primitive marrow cells enhances the expansion of hematopoietic stem cells (HSCs). However, little is known of how expression or function of these proteins is regulated during hematopoiesis under physiological conditions. In our previous studies we demonstrated that thrombopoietin (TPO) enhances levels of HOXB4 mRNA in primitive hematopoietic cells (K. Kirito, N. Fox, and K. Kaushansky, Blood 102:3172-3178, 2003). To extend our studies, we investigated the effects of TPO on HOXA9 in this same cell population. Although overall levels of the transcription factor were not affected, we found that TPO induced the nuclear import of HOXA9 both in UT-7/TPO cells and in primitive Sca-1+/c-kit+/Gr-1− hematopoietic cells in a mitogen-activated protein kinase-dependent fashion. TPO also controlled MEIS1 expression at mRNA levels, at least in part due to phosphatidylinositol 3-kinase activation. Collectively, TPO modulates the function of HOXA9 by leading to its nuclear translocation, likely mediated by effects on its partner protein MEIS1, and potentially due to two newly identified nuclear localization signals. Our data suggest that TPO controls HSC development through the regulation of multiple members of the Hox family of transcription factors through multiple mechanisms.

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JAK/STAT Inhibition Targets TP53 altered Primary Human Acute Myeloid Leukemia Stem Cells

Blood ◽

10.1182/blood-2020-142993 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-28

Author(s):

Marie Lue Antony ◽

Klara Noble-Orcutt ◽

Yoonku Lee ◽

Oluwateniayo Ogunsan ◽

Jeffrey Lee Jensen ◽

...

Keyword(s):

Intracellular Signaling ◽

Expression Profiles ◽

Survival Rates ◽

Gene Expression Profiles ◽

Treatment Resistance ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Poor Risk ◽

Molecular Features

In acute myeloid leukemia (AML), the impact of genetic drivers on response to therapy and long-term survival has been well characterized. AML with complex cytogenetics and TP53 alterations (TP53Alt) is a poor-risk AML subtype that is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplant leading to survival rates 0-10% at 1 year. In contrast, AML with favorable risk molecular features is highly sensitive to chemotherapy and confers survival rates of 50-70%. AML with intermediate risk molecular features can be responsive to chemotherapy and can be cured with hematopoietic stem cell transplant leading to overall survival rates of 30-60%. Leukemia stem cells (LSCs), the cells that recapitulate and propagate leukemia, are central to leukemia progression and relapse. Given the differences in chemo-sensitivity and clinical behavior of genetic subgroups of AML, we asked whether LSCs from poor risk AMLs exhibit distinct signaling activation profiles. We assembled a panel of 23 primary human AML samples with intermediate- and poor- risk genetics and used CyTOF (mass cytometry) to quantitatively measure the levels of immunophenotypic proteins and intracellular signaling molecules in each sample, at the single-cell level. We gated on CD34+CD123+CD3-CD19- cells (LSCs) and measured the level of intracellular signaling molecules within the LSCs of each sample. Notably, the intracellular signaling activation state of LSCs from each AML subtype was distinct; NFkB, pERK, p4EBP1, and pSTAT3 were uniquely upregulated in complex cytogenetics and TP53Alt LSCs, relative to LSCs from intermediate risk AML, suggesting that these signaling pathways may be important for LSC function in this AML subtype. Given that TP53Alt independently confer treatment resistance in AML, we focused on this genetic subgroup. We compared the gene expression profiles of TP53Alt and TP53-wild-type AML samples from the BEAT AML dataset (Tyner et al. Nature 2018) and found that the gene expression profiles of TP53Alt samples are enriched for gene sets representing JAK/STAT signaling, consistent with our CyTOF data, which identified activation of STAT3 in TP53Alt LSCs. A recent drug screen in AML demonstrated that a JAK1/2 kinase inhibitor, AZD1480, can reduce the in vitro viability of TP53-deleted AML cell lines (Nechiporuk et al. Ca Discovery 2019), but these effects were not tested in primary AML samples or on LSCs. Since LSCs confer treatment resistance, we investigated the effect of the AZD1480 on the LSC population in TP53Alt primary human AML samples. AZD1480 treatment abolished all colony formation in primary human TP53Alt AML samples (n=7, 6 replicates per sample, p<0.01). Treatment of these samples in liquid cultures led to a 50% reduction in LSC frequency. We used CyTOF to profile the intracellular signaling states of in vitro treated samples and found that AZD1480 attenuated pSTAT3, pSTAT5, p4EBP1, and NFkB in the LSCs of these samples. The mTOR/4EBp1 and NF༆B pathways have been implicated as drivers of self-renewal and LSC function in AML. Our data suggest that JAK/STAT inhibition may target these pathways in TP53Alt LSCs. These data demonstrate the unique signaling states of TP53Alt LSCs, relative to other LSCs, and show that inhibition of the JAK/STAT pathway specifically targets LSCs within human TP53Alt AML. Figure Disclosures No relevant conflicts of interest to declare.

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