Publications by authors named "Philipp B Staber"

41 Publications

Myeloid lncRNA LOUP Mediates Opposing Regulatory Effects of RUNX1 and RUNX1-ETO in t(8;21) AML.

Blood 2021 05 10. Epub 2021 May 10.

Harvard Medical School Initiative for RNA Medicine, Harvard Medical School, Boston, Massachusetts, United States.

The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA- and DNA-interactions with the broadly expressed transcription factor RUNX1, we identified the long noncoding RNA LOUP. This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia, wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein RUNX1-ETO limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression. These findings highlight the important role of the interplay between cell type-specific RNAs and transcription factors as well as their oncogenic derivatives in modulating lineage-gene activation and raise the possibility that RNA regulators of transcription factors represent alternative targets for therapeutic development.
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http://dx.doi.org/10.1182/blood.2020007920DOI Listing
May 2021

Blood cancer driver Musashi-2 as therapeutic target in chronic lymphocytic leukemia.

Leukemia 2021 04 2;35(4):982-983. Epub 2021 Mar 2.

Division of Hematology and Hemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Austria.

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http://dx.doi.org/10.1038/s41375-021-01144-1DOI Listing
April 2021

BH3 profiling identifies ruxolitinib as a promising partner for venetoclax to treat T-cell prolymphocytic leukemia.

Blood 2021 Feb 17. Epub 2021 Feb 17.

Dana-Farber Cancer Institute, Boston, Massachusetts, United States.

Conventional therapies for patients with T-cell prolymphocytic leukemia (T-PLL) such as cytotoxic chemotherapy and alemtuzumab have limited efficacy and considerable toxicity. Several novel agent classes have demonstrated preclinical activity in T-PLL, including inhibitors of the JAK/STAT and TCR pathways, as well as histone deacetylase (HDAC) inhibitors. Recently, the BCL-2 inhibitor venetoclax also showed some clinical activity in T-PLL. We sought to characterize functional apoptotic dependencies in T-PLL to identify novel combination therapy in this disease. Twenty-four primary T-PLL patient samples were studied using BH3 profiling, a functional assay to assess the propensity of a cell to undergo apoptosis ('priming') and the relative dependence of a cell on different anti-apoptotic proteins. Primary T-PLL cells had a relatively low level of priming for apoptosis, and predominantly depended on BCL-2 and MCL-1 for survival. Selective pharmacologic inhibition of BCL-2 or MCL-1 induced cell death in primary T-PLL cells. Targeting JAK/STAT pathway with the JAK1/2 inhibitor ruxolitinib or HDAC with belinostat both independently increased dependence on BCL-2 but not MCL-1, thereby sensitizing T-PLL cells to venetoclax. Based on these results, we treated two patients with refractory T-PLL with the combination of venetoclax and ruxolitinib. We observed a deep response in the JAK3-mutated T-PLL and a stabilization of the unmutated disease. Our functional, precision medicine-based approach identified inhibitors of HDAC and the JAK/STAT pathway as promising combination partners for venetoclax, warranting further exploration of such combinations clinically in T-PLL.
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http://dx.doi.org/10.1182/blood.2020007303DOI Listing
February 2021

CXCR4 PET imaging of mantle cell lymphoma using [Ga]Pentixafor: comparison with [F]FDG-PET.

Theranostics 2021 1;11(2):567-578. Epub 2021 Jan 1.

Dept. of Biomedical Imaging and Image-guided Therapy, Division of Nuclear Medicine, Medical University of Vienna, Austria.

For PET imaging of mantle cell lymphoma (MCL), [F]FDG (2-deoxy-2-[F]fluoro-D-glucose) is the currently recommended radiotracer, although uptake is variable and bone marrow evaluation is limited. In this prospective study, we evaluated the novel CXCR4 (G-protein-coupled C-X-C chemokine receptor type 4) tracer [Ga]Pentixafor in MCL patients, and compared it to [F]FDG. MCL patients underwent [Ga]Pentixafor-PET/MRI, and, if required for routine purposes, also [F]FDG-PET/MRI, before treatment. PET was evaluated separately for 23 anatomic regions (12 lymph node stations and 11 organs/tissues), using MRI as the main reference standard. Standardized uptake values (SUV and SUV) and tumor-to-background ratios (TBR and TBR) were calculated. General Estimation Equations (GEE) were used to compare [Ga]Pentixafor-PET and [F]FDG-PET sensitivities and positive predictive values (PPV). For bone marrow involvement, where biopsy served as the main reference standard, and splenic involvement, receiver operating characteristic curves were used to determine the optimal SUV and TBR cut-off values, and areas under the curve (AUC) were calculated. Twenty-two MCL patients were included. [Ga]Pentixafor-PET sensitivity (100%) was significantly higher than for [F]FDG-PET (75.2%) (<0.001), and PPV was slightly, but not significantly lower (94.0%.vs. 96.5%; =0.21). SUVs and TBRs were significantly higher for [Ga]Pentixafor-PET than for [F]FDG-PET (<0.001 in all cases); the greatest difference was observed for mean TBR, with 4.9 for [Ga]Pentixafor-PET and 2.0 for [F]FDG-PET. For bone marrow involvement, [Ga]Pentixafor-PET SUV showed an AUC of 0.92; and for splenic involvement, TBR showed an AUC of 0.81. [Ga]Pentixafor-PET may become an alternative to [F]FDG-PET in MCL patients, showing clearly higher detection rates and better tumor-to-background contrast.
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http://dx.doi.org/10.7150/thno.48620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738870PMC
January 2021

Counteracts Anti-Leukemic and Stem Cell Inhibitory Effects of Retinoic Acid on -ITD/-Driven Acute Myeloid Leukemia Cells.

Biomedicines 2020 Sep 28;8(10). Epub 2020 Sep 28.

Division of Oncology, Department of Medicine I, Medical University of Vienna, 1090 Vienna, Austria.

retinoic acid (atRA) has a dramatic impact on the survival of patients with acute promyelocytic leukemia, but its therapeutic value in other types of acute myeloid leukemia (AML) has so far remained unclear. Given that AML is a stem cell-driven disease, recent studies have addressed the effects of atRA on leukemic stem cells (LSCs). atRA promoted stemness of -driven AML in an -dependent manner but had the opposite effect in -ITD/-driven AML. Overexpression of the stem cell-associated transcription factor predicts a poor prognosis in AML, and is observed in different genetic subtypes, including cytogenetically normal AML. Here, we therefore investigated the effects of in a mouse model for cytogenetically normal AML, which rests on the combined activity of -ITD and mutations. Experimental expression of on this background strongly promoted disease aggressiveness. atRA inhibited leukemia cell viability and stem cell-related properties, and these effects were counteracted by overexpression of . These data further underscore the complexity of the responsiveness of AML LSCs to atRA and point out the need for additional investigations which may lay a foundation for a precision medicine-based use of retinoids in AML.
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http://dx.doi.org/10.3390/biomedicines8100385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600968PMC
September 2020

Promotes Aggressiveness and Stem Cell-Related Properties of Acute Myeloid Leukemia.

Cancer Res 2020 10 1;80(20):4527-4539. Epub 2020 Sep 1.

Division of Oncology, Department of Medicine I, Medical University of Vienna, Vienna, Austria.

Overexpression of , which encodes the alpha chain of the IL2 receptor, is associated with chemotherapy resistance and poor outcome in acute myeloid leukemia (AML). The clinical potential of anti-IL2RA therapy is, therefore, being explored in early-stage clinical trials. Notwithstanding, only very limited information regarding the biological function of in AML is available. Using genetic manipulation of expression as well as antibody-mediated inhibition of IL2RA in human cell lines, mouse models, and primary patient samples, we investigated the effects of on AML cell proliferation and apoptosis, and on pertinent signaling pathways. The impact of on the properties of leukemic stem cells (LSC) and on leukemogenesis were queried. promoted proliferation and cell-cycle activity and inhibited apoptosis in human AML cell lines and primary cells. These phenotypes were accompanied by corresponding alterations in cell-cycle machinery and in pathways associated with cell survival and apoptosis. The biological roles of were confirmed in two genetically distinct AML mouse models, revealing that inhibits differentiation, promotes stem cell-related properties, and is required for leukemogenesis. IL2RA antibodies inhibited leukemic, but not normal, hematopoietic cells and synergized with other antileukemic agents in this regard. Collectively, these data show for the first time that plays key biological roles in AML and underscore its value as a potential therapeutic target in this disease. SIGNIFICANCE: This study identifies as a potential therapeutic target in AML, where it is shown to regulate proliferation, differentiation, apoptosis, stem cell-related properties, and leukemogenesis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0531DOI Listing
October 2020

The Bone's Role in Myeloid Neoplasia.

Int J Mol Sci 2020 Jul 1;21(13). Epub 2020 Jul 1.

Division of Hematology and Hemostaseology, Department of Medicine I, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.

The interaction of hematopoietic stem and progenitor cells with their direct neighboring cells in the bone marrow (the so called hematopoietic niche) evolves as a key principle for understanding physiological and malignant hematopoiesis. Significant progress in this matter has recently been achieved making use of emerging high-throughput techniques that allow characterization of the bone marrow microenvironment at single cell resolution. This review aims to discuss these single cell findings in the light of other conventional niche studies that together define the current notion of the niche's implication in i) normal hematopoiesis, ii) myeloid neoplasms and iii) disease-driving pathways that can be exploited to establish novel therapeutic strategies in the future.
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http://dx.doi.org/10.3390/ijms21134712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369750PMC
July 2020

In Human Visualization of Ibrutinib-Induced CLL Compartment Shift.

Cancer Immunol Res 2020 08 24;8(8):984-989. Epub 2020 Jun 24.

Division of Hematology, Department of Medicine I, Medical University of Vienna, Vienna, Austria.

Bruton tyrosine kinase inhibitor ibrutinib is effective in treating chronic lymphocytic leukemia (CLL). However, after ibrutinib treatment initiation, patients frequently experience an increase of CLL blood cell count. This phenomenon in clinical practice is thought to reflect a "compartment shift" of CLL cells from lymph nodes to the peripheral blood, but the actual shifting has not yet been demonstrated. Using [Ga]Pentixafor-PET/MRI for CXCR4 visualization, we here provide images of topical changes of CLL cells upon ibrutinib treatment. Within the first month of ibrutinib treatment, mean standardized [Ga]Pentixafor uptake decreased in the bone marrow and lymph nodes, whereas [Ga]Pentixafor uptake increased in the spleen. Leukocytosis rose, as did numbers of CXCR4 (tissue-resident) CLL cells. Volumes of lymph nodes and spleen decreased. Upon longer ibrutinib treatment, leukocytosis decreased, followed by a decrease of [Ga]Pentixafor uptake in the spleen. These results support the preexisting clinical hypothesis of a "compartment shift" of CLL cells from the lymph nodes to the peripheral blood, but also refine the mechanistic model by describing early clearing of the bone marrow and redistribution of CLL cells to the orthotopic splenic cavernous system in response to ibrutinib treatment.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0880DOI Listing
August 2020

Targeting Nuclear NOTCH2 by Gliotoxin Recovers a Tumor-Suppressor NOTCH3 Activity in CLL.

Cells 2020 06 18;9(6). Epub 2020 Jun 18.

Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, 1090 Vienna, Austria.

NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the (CD23) axis together with concomitant upregulation of the axis. In contrast, RO4929097 downregulated the axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.
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http://dx.doi.org/10.3390/cells9061484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348714PMC
June 2020

UGT2B17 modifies drug response in chronic lymphocytic leukaemia.

Br J Cancer 2020 07 18;123(2):240-251. Epub 2020 May 18.

Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHU de Québec) Research Center and Faculty of Pharmacy, Laval University, Québec, QC, Canada.

Background: High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents.

Methods: Functional enzymatic assays and patients' plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels.

Results: High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub.

Conclusions: Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17.
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http://dx.doi.org/10.1038/s41416-020-0887-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374097PMC
July 2020

[18F]FDG-PET/CT Radiomics for Prediction of Bone Marrow Involvement in Mantle Cell Lymphoma: A Retrospective Study in 97 Patients.

Cancers (Basel) 2020 05 2;12(5). Epub 2020 May 2.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Biopsy is the standard for assessment of bone marrow involvement in mantle cell lymphoma (MCL). We investigated whether [18F]FDG-PET radiomic texture features can improve prediction of bone marrow involvement in MCL, compared to standardized uptake values (SUV), and whether combination with laboratory data improves results. Ninety-seven MCL patients were retrospectively included. SUVmax, SUVmean, SUVpeak and 16 co-occurrence matrix texture features were extracted from pelvic bones on [18F]FDG-PET/CT. A multi-layer perceptron neural network was used to compare three combinations for prediction of bone marrow involvement-the SUVs, a radiomic signature based on SUVs and texture features, and the radiomic signature combined with laboratory parameters. This step was repeated using two cut-off values for relative bone marrow involvement: REL > 5% (>5% of red/cellular bone marrow); and REL > 10%. Biopsy demonstrated bone marrow involvement in 67/97 patients (69.1%). SUVs, the radiomic signature, and the radiomic signature with laboratory data showed AUCs of up to 0.66, 0.73, and 0.81 for involved vs. uninvolved bone marrow; 0.68, 0.84, and 0.84 for REL ≤ 5% vs. REL > 5%; and 0.69, 0.85, and 0.87 for REL ≤ 10% vs. REL > 10%. In conclusion, [18F]FDG-PET texture features improve SUV-based prediction of bone marrow involvement in MCL. The results may be further improved by combination with laboratory parameters.
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http://dx.doi.org/10.3390/cancers12051138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281173PMC
May 2020

RECIL versus Lugano for Treatment Response Assessment in FDG-Avid Non-Hodgkin Lymphomas: A Head-to-Head Comparison in 54 Patients.

Cancers (Basel) 2019 Dec 18;12(1). Epub 2019 Dec 18.

Department of Biomedical Imaging and Image-guided Therapy, Division of General and Pediatric Radiology, Medical University of Vienna, 1090 Vienna, Austria.

The response evaluation criteria in lymphoma (RECIL) classification for lymphoma treatment response assessment was introduced in 2017, but it has not yet been compared to the established Lugano classification. Also, the value of the provisional "minor response" (MiR) category of RECIL is unclear. In 54 patients with FDG-avid non-Hodgkin lymphomas (41 diffuse large B-cell lymphomas (DLBCL) and 13 follicular lymphomas), [F]FDG-PET/CT-based response according to RECIL and Lugano was determined at interim and end-of-treatment (EOT) restaging. Rates of agreement and Cohen's kappa (κ) coefficients were calculated. The relationship between RECIL and Lugano responses and 2-year complete remission (CR) status of DLBCL patients was determined. At interim restaging, MiR was observed in 14.8%, and at EOT, in 5.6% of patients. When MiR was recoded as partial remission, agreement between RECIL and Lugano was 83.3% at interim restaging (κ = 0.69), and 90.7% at EOT (κ = 0.79). 85.4%, of DLBCL patients with responding disease at interim restaging according to both RECIL and Lugano achieved 2-year CR status; whereas, at EOT, 82.9% of patients with responding disease according to Lugano, and 85.4% of patients with responding disease according to RECIL, achieved 2-year CR status. Thus, RECIL and Lugano classifications show comparable performance for treatment response assessment, and a similar association with 2-year CR status in FDG-avid lymphomas.
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http://dx.doi.org/10.3390/cancers12010009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016710PMC
December 2019

All-trans retinoic acid enhances, and a pan-RAR antagonist counteracts, the stem cell promoting activity of EVI1 in acute myeloid leukemia.

Cell Death Dis 2019 12 10;10(12):944. Epub 2019 Dec 10.

Division of Oncology, Clinic of Medicine I, Medical University of Vienna, Vienna, Austria.

Ecotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1 AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1 AML.
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http://dx.doi.org/10.1038/s41419-019-2172-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904467PMC
December 2019

Consensus criteria for diagnosis, staging, and treatment response assessment of T-cell prolymphocytic leukemia.

Blood 2019 10 10;134(14):1132-1143. Epub 2019 Jul 10.

The Royal Marsden Hospital, NHS Foundation Trust, London, United Kingdom.

T-cell prolymphocytic leukemia (T-PLL) is a rare, mature T-cell neoplasm with a heterogeneous clinical course. With the advent of novel treatment options that will potentially change the management of patients with T-PLL, it has become necessary to produce consensus guidelines for the design and conduct of clinical trials. The T-PLL International Study group (TPLL-ISG) set out to define standardized criteria for diagnosis, treatment indication, and evaluation of response. These criteria will facilitate comparison of results from clinical trials in T-PLL, and will thus support clinical decision making, as well as the approval of new therapeutics by healthcare authorities.
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http://dx.doi.org/10.1182/blood.2019000402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042666PMC
October 2019

Combined chemosensitivity and chromatin profiling prioritizes drug combinations in CLL.

Nat Chem Biol 2019 03 28;15(3):232-240. Epub 2019 Jan 28.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has substantially improved therapeutic options for chronic lymphocytic leukemia (CLL). Although ibrutinib is not curative, it has a profound effect on CLL cells and may create new pharmacologically exploitable vulnerabilities. To identify such vulnerabilities, we developed a systematic approach that combines epigenome profiling (charting the gene-regulatory basis of cell state) with single-cell chemosensitivity profiling (quantifying cell-type-specific drug response) and bioinformatic data integration. By applying our method to a cohort of matched patient samples collected before and during ibrutinib therapy, we identified characteristic ibrutinib-induced changes that provide a starting point for the rational design of ibrutinib combination therapies. Specifically, we observed and validated preferential sensitivity to proteasome, PLK1, and mTOR inhibitors during ibrutinib treatment. More generally, our study establishes a broadly applicable method for investigating treatment-specific vulnerabilities by integrating the complementary perspectives of epigenetic cell states and phenotypic drug responses in primary patient samples.
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http://dx.doi.org/10.1038/s41589-018-0205-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746620PMC
March 2019

Dependency on the TYK2/STAT1/MCL1 axis in anaplastic large cell lymphoma.

Leukemia 2019 03 21;33(3):696-709. Epub 2018 Aug 21.

Cancer Science Institute of Singapore, National University of Singapore, 117599, Singapore, Singapore.

TYK2 is a member of the JAK family of tyrosine kinases that is involved in chromosomal translocation-induced fusion proteins found in anaplastic large cell lymphomas (ALCL) that lack rearrangements activating the anaplastic lymphoma kinase (ALK). Here we demonstrate that TYK2 is highly expressed in all cases of human ALCL, and that in a mouse model of NPM-ALK-induced lymphoma, genetic disruption of Tyk2 delays the onset of tumors and prolongs survival of the mice. Lymphomas in this model lacking Tyk2 have reduced STAT1 and STAT3 phosphorylation and reduced expression of Mcl1, a pro-survival member of the BCL2 family. These findings in mice are mirrored in human ALCL cell lines, in which TYK2 is activated by autocrine production of IL-10 and IL-22 and by interaction with specific receptors expressed by the cells. Activated TYK2 leads to STAT1 and STAT3 phosphorylation, activated expression of MCL1 and aberrant ALCL cell survival. Moreover, TYK2 inhibitors are able to induce apoptosis in ALCL cells, regardless of the presence or absence of an ALK-fusion. Thus, TYK2 is a dependency that is required for ALCL cell survival through activation of MCL1 expression. TYK2 represents an attractive drug target due to its essential enzymatic domain, and TYK2-specific inhibitors show promise as novel targeted inhibitors for ALCL.
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http://dx.doi.org/10.1038/s41375-018-0239-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076043PMC
March 2019

CD44 is a RAS/STAT5-regulated invasion receptor that triggers disease expansion in advanced mastocytosis.

Blood 2018 11 17;132(18):1936-1950. Epub 2018 Jul 17.

Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria.

The Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and invasion of neoplastic stem cells in various myeloid malignancies. Although mast cells (MCs) reportedly express CD44, little is known about the regulation and function of this receptor in neoplastic cells in systemic mastocytosis (SM). We found that clonal CD34/CD38 stem cells, CD34/CD38 progenitor cells, and CD117/CD34 MCs invariably express CD44 in patients with indolent SM (ISM), SM with an associated hematologic neoplasm, aggressive SM, and MC leukemia (MCL). In addition, all human MCL-like cell lines examined (HMC-1, ROSA, and MCPV-1) displayed cytoplasmic and cell-surface CD44. We also found that expression of CD44 in neoplastic MCs depends on RAS-MEK and STAT5 signaling and increases with the aggressiveness of SM. Correspondingly, higher levels of soluble CD44 were measured in the sera of patients with advanced SM compared with ISM or cutaneous mastocytosis and were found to correlate with overall and progression-free survival. To investigate the functional role of CD44, a xenotransplantation model was employed using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and a short hairpin RNA (shRNA) against CD44. In this model, the shRNA-mediated knockdown of CD44 resulted in reduced MC expansion and tumor formation and prolonged survival in SCID mice compared with HMC-1.2 cells transduced with control shRNA. Together, our data show that CD44 is a RAS-MEK/STAT5-driven MC invasion receptor that correlates with the aggressiveness of SM. Whether CD44 can serve as therapeutic target in advanced SM remains to be determined in forthcoming studies.
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http://dx.doi.org/10.1182/blood-2018-02-833582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382065PMC
November 2018

Proposed Terminology and Classification of Pre-Malignant Neoplastic Conditions: A Consensus Proposal.

EBioMedicine 2017 Dec 26;26:17-24. Epub 2017 Nov 26.

Institute of Pathology, Ludwig-Maximilian University, Munich, Germany.

Cancer evolution is a step-wise non-linear process that may start early in life or later in adulthood, and includes pre-malignant (indolent) and malignant phases. Early somatic changes may not be detectable or are found by chance in apparently healthy individuals. The same lesions may be detected in pre-malignant clonal conditions. In some patients, these lesions may never become relevant clinically whereas in others, they act together with additional pro-oncogenic hits and thereby contribute to the formation of an overt malignancy. Although some pre-malignant stages of a malignancy have been characterized, no global system to define and to classify these conditions is available. To discuss open issues related to pre-malignant phases of neoplastic disorders, a working conference was organized in Vienna in August 2015. The outcomes of this conference are summarized herein and include a basic proposal for a nomenclature and classification of pre-malignant conditions. This proposal should assist in the communication among patients, physicians and scientists, which is critical as genome-sequencing will soon be offered widely for early cancer-detection.
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http://dx.doi.org/10.1016/j.ebiom.2017.11.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832623PMC
December 2017

Image-based ex-vivo drug screening for patients with aggressive haematological malignancies: interim results from a single-arm, open-label, pilot study.

Lancet Haematol 2017 Dec 15;4(12):e595-e606. Epub 2017 Nov 15.

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria; Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria.

Background: Patients with refractory or relapsed haematological malignancies have few treatment options and short survival times. Identification of effective therapies with genomic-based precision medicine is hampered by intratumour heterogeneity and incomplete understanding of the contribution of various mutations within specific cancer phenotypes. Ex-vivo drug-response profiling in patient biopsies might aid effective treatment identification; however, proof of its clinical utility is limited.

Methods: We investigated the feasibility and clinical impact of multiparametric, single-cell, drug-response profiling in patient biopsies by immunofluorescence, automated microscopy, and image analysis, an approach we call pharmacoscopy. First, the ability of pharmacoscopy to separate responders from non-responders was evaluated retrospectively for a cohort of 20 newly diagnosed and previously untreated patients with acute myeloid leukaemia. Next, 48 patients with aggressive haematological malignancies were prospectively evaluated for pharmacoscopy-guided treatment, of whom 17 could receive the treatment. The primary endpoint was progression-free survival in pharmacoscopy-treated patients, as compared with their own progression-free survival for the most recent regimen on which they had progressive disease. This trial is ongoing and registered with ClinicalTrials.gov, number NCT03096821.

Findings: Pharmacoscopy retrospectively predicted the clinical response of 20 acute myeloid leukaemia patients to initial therapy with 88·1% accuracy. In this interim analysis, 15 (88%) of 17 patients receiving pharmacoscopy-guided treatment had an overall response compared with four (24%) of 17 patients with their most recent regimen (odds ratio 24·38 [95% CI 3·99-125·4], p=0·0013). 12 (71%) of 17 patients had a progression-free survival ratio of 1·3 or higher, and median progression-free survival increased by four times, from 5·7 (95% CI 4·1-12·1) weeks to 22·6 (7·4-34·0) weeks (hazard ratio 3·14 [95% CI 1·37-7·22], p=0·0075).

Interpretation: Routine clinical integration of pharmacoscopy for treatment selection is technically feasible, and led to improved treatment of patients with aggressive refractory haematological malignancies in an initial patient cohort, warranting further investigation.

Funding: Austrian Academy of Sciences; European Research Council; Austrian Science Fund; Austrian Federal Ministry of Science, Research and Economy; National Foundation for Research, Technology and Development; Anniversary Fund of the Austrian National Bank; MPN Research Foundation; European Molecular Biology Organization; and Swiss National Science Foundation.
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http://dx.doi.org/10.1016/S2352-3026(17)30208-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719985PMC
December 2017

First-in-human response of BCL-2 inhibitor venetoclax in T-cell prolymphocytic leukemia.

Blood 2017 12 27;130(23):2499-2503. Epub 2017 Sep 27.

Division of Hematology and Hemostaseology, Department of Internal Medicine I.

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive T-lymphoid malignancy usually refractory to current treatment strategies and associated with short overall survival. By applying next-generation functional testing of primary patient-derived lymphoma cells using a library of 106 US Food and Drug Administration (FDA)-approved anticancer drugs or compounds currently in clinical development, we set out to identify novel effective treatments for T-PLL patients. We found that the B-cell lymphoma 2 (BCL-2) inhibitor venetoclax (ABT-199) demonstrated the strongest T-PLL-specific response when comparing individual ex vivo drug response in 86 patients with refractory hematologic malignancies. Mechanistically, responses to venetoclax correlated with protein expression of BCL-2 but not with expression of the BCL-2 family members myeloid cell leukemia 1 (MCL-1) and BCL-XL in lymphoma cells. BCL-2 expression was inversely correlated with the expression of MCL-1. Based on the ex vivo responses, venetoclax treatment was commenced in 2 late-stage refractory T-PLL patients resulting in clinical responses. Our findings demonstrate first evidence of single-agent activity of venetoclax both ex vivo and in humans, offering a novel agent in T-PLL.
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http://dx.doi.org/10.1182/blood-2017-05-785683DOI Listing
December 2017

When the guardian sleeps: Reactivation of the p53 pathway in cancer.

Mutat Res 2017 07 17;773:1-13. Epub 2017 Feb 17.

Department of Clinical Pathology, Medical University Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria; Ludwig Boltzmann Institute for Cancer Research, Waehringerstrasse 13a, 1090 Vienna, Austria; Institute of Laboratory Animal Pathology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, Vienna, Austria. Electronic address:

The p53 tumor suppressor is inactivated in most cancers, thus suggesting that loss of p53 is a prerequisite for tumor growth. Therefore, its reintroduction through different means bears great clinical potential. After a brief introduction to current knowledge of p53 and its regulation by the ubiquitin-ligases MDM2/MDMX and post-translational modifications, we will discuss small molecules that are able to reactivate specific, frequently observed mutant forms of p53 and their applicability for clinical purposes. Many malignancies display amplification of MDM genes encoding negative regulators of p53 and therefore much effort to date has concentrated on the development of molecules that inhibit MDM2, the most advanced of which are being tested in clinical trials for sarcoma, glioblastoma, bladder cancer and lung adenocarcinoma. These will be discussed as will recent findings of MDMX inhibitors: these are of special importance as it has been shown that cancers that become resistant to MDM2 inhibitors often amplify MDM4. Finally, we will also touch on gene therapy and vaccination approaches; the former of which aims to replace mutated TP53 and the latter whose goal is to activate the body's immune system toward mutant p53 expressing cells. Besides the obvious importance of MDM2 and MDMX expression for regulation of p53, other regulatory factors should not be underestimated and are also described. Despite the beauty of the concept, the past years have shown that many obstacles have to be overcome to bring p53 reactivation to the clinic on a broad scale, and it is likely that in most cases it will be part of a combined therapeutic approach. However, improving current p53 targeted molecules and finding the best therapy partners will clearly impact the future of cancer therapy.
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http://dx.doi.org/10.1016/j.mrrev.2017.02.003DOI Listing
July 2017

DNMT3A mutations promote anthracycline resistance in acute myeloid leukemia via impaired nucleosome remodeling.

Nat Med 2016 12 14;22(12):1488-1495. Epub 2016 Nov 14.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3A), have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3A AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy, suggesting that DNMT3A cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3) and the nucleophosmin gene (Npm1) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3A mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3A cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3A mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.
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http://dx.doi.org/10.1038/nm.4210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359771PMC
December 2016

The DNA Ligase IV Syndrome R278H Mutation Impairs B Lymphopoiesis via Error-Prone Nonhomologous End-Joining.

J Immunol 2016 Jan 25;196(1):244-55. Epub 2015 Nov 25.

Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215; Broad Institute of MIT and Harvard, Cambridge, MA 02142; and Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115

Hypomorphic mutations in the nonhomologous end-joining (NHEJ) DNA repair protein DNA ligase IV (LIG4) lead to immunodeficiency with varying severity. In this study, using a murine knock-in model, we investigated the mechanisms underlying abnormalities in class switch recombination (CSR) associated with the human homozygous Lig4 R278H mutation. Previously, we found that despite the near absence of Lig4 end-ligation activity and severely reduced mature B cell numbers, Lig4(R278H/R278H) (Lig4(R/R)) mice exhibit only a partial CSR block, producing near normal IgG1 and IgE but substantially reduced IgG3, IgG2b, and IgA serum levels. In this study, to address the cause of these abnormalities, we assayed CSR in Lig4(R/R) B cells generated via preassembled IgH and IgK V region exons (HL). This revealed that Lig4(R278H) protein levels while intact exhibited a higher turnover rate during activation of switching to IgG3 and IgG2b, as well as delays in CSR kinetics associated with defective proliferation during activation of switching to IgG1 and IgE. Activated Lig4(R/R)HL B cells consistently accumulated high frequencies of activation-induced cytidine deaminase-dependent IgH locus chromosomal breaks and translocations and were more prone to apoptosis, effects that appeared to be p53-independent, as p53 deficiency did not markedly influence these events. Importantly, NHEJ instead of alternative end-joining (A-EJ) was revealed as the predominant mechanism catalyzing robust CSR. Defective CSR was linked to failed NHEJ and residual A-EJ access to unrepaired double-strand breaks. These data firmly demonstrate that Lig4(R278H) activity renders NHEJ to be more error-prone, and they predict increased error-prone NHEJ activity and A-EJ suppression as the cause of the defective B lymphopoiesis in Lig4 patients.
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http://dx.doi.org/10.4049/jimmunol.1403099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684978PMC
January 2016

Hematopoietic Differentiation Is Required for Initiation of Acute Myeloid Leukemia.

Cell Stem Cell 2015 Nov 24;17(5):611-23. Epub 2015 Sep 24.

Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA; Cancer Science Institute, National University of Singapore, Singapore, 117599. Electronic address:

Mutations in acute myeloid leukemia (AML)-associated oncogenes often arise in hematopoietic stem cells (HSCs) and promote acquisition of leukemia stem cell (LSC) phenotypes. However, as LSCs often share features of lineage-restricted progenitors, the relative contribution of differentiation status to LSC transformation is unclear. Using murine MLL-AF9 and MOZ-TIF2 AML models, we show that myeloid differentiation to granulocyte macrophage progenitors (GMPs) is critical for LSC generation. Disrupting GMP formation by deleting the lineage-restricted transcription factor C/EBPa blocked normal granulocyte formation and prevented initiation of AML. However, restoring myeloid differentiation in C/EBPa mutants with inflammatory cytokines reestablished AML transformation capacity. Genomic analyses of GMPs, including gene expression and H3K79me2 profiling in conjunction with ATAC-seq, revealed a permissive genomic environment for activation of a minimal transcription program shared by GMPs and LSCs. Together, these findings show that myeloid differentiation is a prerequisite for LSC formation and AML development, providing insights for therapeutic development.
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http://dx.doi.org/10.1016/j.stem.2015.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636971PMC
November 2015

The Runx-PU.1 pathway preserves normal and AML/ETO9a leukemic stem cells.

Blood 2014 Oct 3;124(15):2391-9. Epub 2014 Sep 3.

Harvard Stem Cell Institute, Harvard Medical School, Boston, MA; Cancer Science Institute, National University of Singapore, Singapore; and.

Runx transcription factors contribute to hematopoiesis and are frequently implicated in hematologic malignancies. All three Runx isoforms are expressed at the earliest stages of hematopoiesis; however, their function in hematopoietic stem cells (HSCs) is not fully elucidated. Here, we show that Runx factors are essential in HSCs by driving the expression of the hematopoietic transcription factor PU.1. Mechanistically, by using a knockin mouse model in which all three Runx binding sites in the -14kb enhancer of PU.1 are disrupted, we observed failure to form chromosomal interactions between the PU.1 enhancer and its proximal promoter. Consequently, decreased PU.1 levels resulted in diminished long-term HSC function through HSC exhaustion, which could be rescued by reintroducing a PU.1 transgene. Similarly, in a mouse model of AML/ETO9a leukemia, disrupting the Runx binding sites resulted in decreased PU.1 levels. Leukemia onset was delayed, and limiting dilution transplantation experiments demonstrated functional loss of leukemia-initiating cells. This is surprising, because low PU.1 levels have been considered a hallmark of AML/ETO leukemia, as indicated in mouse models and as shown here in samples from leukemic patients. Our data demonstrate that Runx-dependent PU.1 chromatin interaction and transcription of PU.1 are essential for both normal and leukemia stem cells.
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http://dx.doi.org/10.1182/blood-2014-01-550855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192750PMC
October 2014

Sox4 is a key oncogenic target in C/EBPα mutant acute myeloid leukemia.

Cancer Cell 2013 Nov 31;24(5):575-88. Epub 2013 Oct 31.

Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02215, USA; Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.

Mutation or epigenetic silencing of the transcription factor C/EBPα is observed in ∼10% of patients with acute myeloid leukemia (AML). In both cases, a common global gene expression profile is observed, but downstream targets relevant for leukemogenesis are not known. Here, we identify Sox4 as a direct target of C/EBPα whereby its expression is inversely correlated with C/EBPα activity. Downregulation of Sox4 abrogated increased self-renewal of leukemic cells and restored their differentiation. Gene expression profiles of leukemia-initiating cells (LICs) from both Sox4 overexpression and murine C/EBPα mutant AML models clustered together but differed from other types of AML. Our data demonstrate that Sox4 overexpression resulting from C/EBPα inactivation contributes to the development of leukemia with a distinct LIC phenotype.
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http://dx.doi.org/10.1016/j.ccr.2013.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038627PMC
November 2013

C/EBPa controls acquisition and maintenance of adult haematopoietic stem cell quiescence.

Nat Cell Biol 2013 Apr 17;15(4):385-94. Epub 2013 Mar 17.

Harvard Stem Cell Institute, Harvard Medical School, and Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115, USA.

In blood, the transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver haematopoietic stem cells (HSCs). However, its function in adult HSCs has remained unknown. Here, using an inducible knockout model we found that C/EBPa-deficient adult HSCs underwent a pronounced increase in number with enhanced proliferation, characteristics resembling fetal liver HSCs. Consistently, transcription profiling of C/EBPa-deficient HSCs revealed a gene expression program similar to fetal liver HSCs. Moreover, we observed that age-specific Cebpa expression correlated with its inhibitory effect on the HSC cell cycle. Mechanistically we identified N-Myc as a downstream target of C/EBPa, and loss of C/EBPa resulted in de-repression of N-Myc. Our data establish C/EBPa as a central determinant in the switch from fetal to adult HSCs.
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http://dx.doi.org/10.1038/ncb2698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781213PMC
April 2013

Sustained PU.1 levels balance cell-cycle regulators to prevent exhaustion of adult hematopoietic stem cells.

Mol Cell 2013 Mar 8;49(5):934-46. Epub 2013 Feb 8.

Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA.

To provide a lifelong supply of blood cells, hematopoietic stem cells (HSCs) need to carefully balance both self-renewing cell divisions and quiescence. Although several regulators that control this mechanism have been identified, we demonstrate that the transcription factor PU.1 acts upstream of these regulators. So far, attempts to uncover PU.1's role in HSC biology have failed because of the technical limitations of complete loss-of-function models. With the use of hypomorphic mice with decreased PU.1 levels specifically in phenotypic HSCs, we found reduced HSC long-term repopulation potential that could be rescued completely by restoring PU.1 levels. PU.1 prevented excessive HSC division and exhaustion by controlling the transcription of multiple cell-cycle regulators. Levels of PU.1 were sustained through autoregulatory PU.1 binding to an upstream enhancer that formed an active looped chromosome architecture in HSCs. These results establish that PU.1 mediates chromosome looping and functions as a master regulator of HSC proliferation.
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http://dx.doi.org/10.1016/j.molcel.2013.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644723PMC
March 2013