Chronic myelomonocytic leukemia (CMML) is a stem cell-derived hematopoietic neoplasm characterized by dysplasia, uncontrolled expansion of monocytic (progenitor) cells in the bone marrow (BM) and in ...the peripheral blood (PB), and an increased risk of progression to secondary acute myeloid leukemia (sAML). Patients with advanced CMML and sAML are often highly resistant to therapy and their prognosis is dismal. It is thought that drug resistance in myeloid malignancies is a quality of leukemia stem cells (LSC), but little is known about CMML-initiating and propagating LSC. We investigated the phenotype and functional behavior of putative CMML-initiating cells in 15 patients with CMML (7 females, 8 males; median age 73 years; range 45-82 years) and 6 with sAML following CMML (1 female, 5 males; median age 67.5 years; range 66-76 years). BM and/or PB samples were examined by multicolor flow cytometry using antibodies against CD34, CD38 and various additional surface markers and target antigens. In a subset of patients, putative stem and progenitor cells (CD34+ cells, CD34+/CD38─ cells and CD34+/CD38+ cells) were FACS-sorted to high purity (>95%) and were employed in xenotransplantation experiments or in drug testing experiments. We found that CMML-initiating and propagating LSC reside within the CD34+/CD38─ fraction of the malignant clone. Whereas highly purified CD34+ cells engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG) 1Eav/MloySzJ (NSGS) mice with full-blown CMML (engraftment rate 44.8±26.0%), no CMML was produced by the bulk of CD34- monocytic cells (engraftment rate 0.8±0.5%; p=0.002). CMML engraftment was also detectable when transplanting unselected mononuclear cells (engraftment rate 19.7±10.9%). By contrast, no leukemic engraftment was produced by CD38+ CMML fractions (engraftment rate 0.1±0.1%; p=0.003), indicating that the NSGS-repopulating CMML LSC reside specifically in a CD34+/CD38- fraction of the clone. In sAML, both the CD34+/CD38- cell fraction (engraftment rate 92.2±6.2%) and the CD34+/CD38+ fraction (engraftment rate 80.5±7.2%) produced engraftment with AML blasts in NSGS mice. In a next step, we established the cell surface phenotype of CD34+/CD38- LSC in CMML and sAML. As assessed by multicolor flow cytometry, CD34+/CD38- CMML cells invariably expressed CD33/Siglec-3, CD117/KIT, CD123/IL3RA, CD133/AC133, CD135/FLT3, and IL-1RAP. In a subset of patients, CMML LSC also expressed CD52 (9/11 patients; 81%), CD114 (3/7 patients; 43%), CD184 (9/12 patients; 75%), CD221 (8/11 patients; 73%) and/or CLL-1 (7/13 patients; 54%). CMML LSC did not express CD25 or CD26. However, in patients with sAML, LSC also displayed CD25 (median fluorescence intensity, MFI: CMML: 0.9 vs. sAML: 23.0; p<0.001). Compared to hematopoietic stem cells in normal BM (NBM), CMML LSC displayed slightly increased levels of CD117/KIT (MFI CMML: 32.5 vs. MFI NBM: 15.0; p=0.019), CD135/FLT3 (MFI CMML: 1.9 vs. MFI NBM: 0.8; p=0.001), CD184/CXCR4 (MFI CMML: 1.6 vs. MFI NBM 0.9; p=0.027), and IL-1RAP (MFI CMML: 1.6 vs. MFI NBM: 0.8; p=0.004). No correlations between surface-marker expression on LSC and the type of CMML (CMML-0/1/2 or dysplastic vs. proliferative CMML) or the clinical course were found. To confirm the clinical relevance of expression of surface target antigens on CMML LSC, we applied the CD33-targeted drug gemtuzumab-ozogamicin (GO). As assessed by combined staining for LSC (CD34+/CD38-) and AnnexinV/DAPI, incubation of CMML LSC with GO (0.001-1 µg/ml) resulted in dose-dependent apoptosis in all donors tested, and the same result was obtained in the monoblastic cell lines THP-1 (GO at 1 µg/ml: 94.2±1.5% vs. control: 12.7±2.2%, p<0.05) and Mono-Mac-1 (GO at 1 µg/ml: 56.4±12.1% vs. control: 10.1±0.6% p<0.05). In conclusion, LSC in CMML and sAML reside within CD34+/CD38─ cell populations that express distinct profiles of surface markers and target antigens. During progression of CMML into sAML, LSC apparently acquire CD25. Characterization of CMML LSC and LSC in sAML should facilitate their enrichment and the development of LSC-eradicating therapies.
Hoermann:Novartis: Honoraria; Roche: Honoraria. Sperr:Celgene: Consultancy, Honoraria; Novartis: Honoraria. Sill:Astex: Other: Advisory board; Novartis: Other: Advisory board; AbbVie: Other: Advisory board; Astellas: Other: Advisory board. Geissler:Novartis: Honoraria; AOP: Honoraria; Roche: Honoraria; Amgen: Honoraria; AstraZeneca: Honoraria; Ratiopharm: Honoraria; Celgene: Honoraria; Abbvie: Honoraria; Pfizer: Honoraria. Deininger:Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Ascentage Pharma: Consultancy, Honoraria; Fusion Pharma: Consultancy; TRM: Consultancy; Sangoma: Consultancy; Adelphi: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy. Valent:Pfizer: Honoraria; Blueprint: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Deciphera: Honoraria, Research Funding.
Chronic myeloid leukemia (CML) is a hematopoietic stem cell neoplasm in which BCR-ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all ...patients, leukemic cells can be kept under control by BCR-ABL1 tyrosine kinase inhibitors (TKI). Nevertheless, resistance against one or more TKI may occur. Therefore, research is focusing on novel potential drug targets in CML. We have recently identified the epigenetic reader bromodomain-containing protein 4 (BRD4) as a new therapeutic target in leukemic stem cells (LSC) in acute myeloid leukemia. In the present study, we examine the expression of BRD4 and its downstream effector MYC in CML cells and asked whether BRD4 serves as a drug target in CML cells and whether BRD4-targeting drugs, including JQ1 and newly developed BRD4 degraders (dBET1 and dBET6) are able to overcome LSC resistance in CML. Primary CML cells were obtained from 22 patients with chronic phase (CP) CML and 3 with blast phase (BP) CML. As determined by qPCR and/or immunocytochemistry, the CML cell lines KU812 and K562 as well as primary CML cells expressed BRD4 and MYC. All three BRD4-targeting drugs (JQ1, dBET1 and dBET6) were found to decrease MYC expression in KU812 and K562 cells as assessed by Western blotting. In 3H-thymidine uptake experiments, JQ1 and dBET6 were found to inhibit the proliferation of KU812 in a dose-dependent manner (IC50, JQ1: 100-500 nM; dBET6: 50-100 nM) whereas dBET1 showed only little if any effects on growth of KU812 cells (IC50: 1-5 µM), and in K562 cells, only dBET6 was found to inhibit growth with a reasonable IC50 value (250-500 nM). Corresponding results were obtained when examining drug effects on survival of CML cell lines by Annexin-V/PI staining. All three BRD4-targeting drugs were found to inhibit proliferation of primary CP CML cells with varying IC50 values. As expected, growth-inhibitory effects of dBET6 were more pronounced (IC50: <100 nM) compared to effects seen with JQ1 and dBET1. dBET1 and dBET6 were also found to inhibit growth of primary CML cells obtained from patients with BP CML, whereas JQ1 was not effective. JQ1 also failed to suppress survival on CML CD34+/CD38− LSC. By contrast, dBET1 induced apoptosis in CML LSC at 1 µM and dBET6 induced apoptosis in CML LSC at 0.1 µM. dBET6 induced apoptosis in CML LSC obtained from patients with imatinib-sensitive CML as well as patients with imatinib-resistant CML harboring BCR-ABL1 T315I or BCR-ABL1 F317L. Finally, pre-incubation of CD34+ CP CML cells with dBET6 resulted in reduced leukemic engraftment in NSG mice exhibiting human membrane-bound stem cell factor, SCF NSG-Tg(hu-mSCF) 6 months after transplantation (engraftment with CD45+/CD33+/CD19−cells in control mice receiving DMSO-treated cells: 8.1±6.6% vs mice receiving dBET6-treated cells: 1.1±0.6%). To further explore the ability of dBET6 to interfere with LSC resistance in CML, we established a co-culture system mimicking LSC-niche interactions in the osteoblastic niche. In this model, co-culturing K562 cells, KU812 cells or primary CML LSC with the osteoblast-like osteosarcoma cell line CAL-72 resulted in resistance against nilotinib and ponatinib. In this culture system, JQ1 was found to partially restore TKI effects in K562 cells and completely restored TKI effects in KU812 cells. Interestingly, JQ1 was not able to restore TKI effects in primary CML LSC in these co-cultures. However, dBET6 was found to overcome niche cell-induced TKI-resistance of primary CML LSC. Finally, we were able to demonstrate that JQ1, dBET1 and dBET6 inhibit interferon-gamma-induced upregulation of PD-L1 expression in CML LSC. Together we show that BRD4 and MYC are potential new therapeutic drug targets in CML and that the BET-degrader dBET6 overcomes multiple forms of LSC resistance, including i) intrinsic resistance, ii) mutation-induced resistance, iii) niche induced resistance and iv) checkpoint-mediated resistance. Whether BRD4 degradation is also able to overcome TKI-resistance of BCR-ABL1+ LSC in vivo in patients with CML remains to be determined in clinical trials.
Hoermann:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria. Wolf:BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding. Mayer:Amgen: Research Funding; Novartis: Research Funding. Zuber:Mirimus Inc.: Consultancy, Other: Shareholder; Boehringer Ingelheim GmbH & Co KG: Research Funding. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Valent:Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria.
The CD52-targeted antibody alemtuzumab induces major clinical responses in a group of patients with myelodysplastic syndromes (MDS). The mechanism underlying this drug effect remains unknown.
We ...asked whether neoplastic stem cells (NSC) in patients with MDS (n = 29) or acute myelogenous leukemia (AML; n = 62) express CD52.
As assessed by flow cytometry, CD52 was found to be expressed on NSC-enriched CD34(+)/CD38(-) cells in 8/11 patients with MDS and isolated del(5q). In most other patients with MDS, CD52 was weakly expressed or not detectable on NSC. In AML, CD34(+)/CD38(-) cells displayed CD52 in 23/62 patients, including four with complex karyotype and del(5q) and one with del(5q) and t(1;17;X). In quantitative PCR (qPCR) analyses, purified NSC obtained from del(5q) patients expressed CD52 mRNA. We were also able to show that CD52 mRNA levels correlate with EVI1 expression and that NRAS induces the expression of CD52 in AML cells. The CD52-targeting drug alemtuzumab, was found to induce complement-dependent lysis of CD34(+)/CD38(-)/CD52(+) NSC, but did not induce lysis in CD52(-) NSC. Alemtuzumab also suppressed engraftment of CD52(+) NSC in NSG mice. Finally, CD52 expression on NSC was found to correlate with a poor survival in patients with MDS and AML.
The cell surface target Campath-1 (CD52) is expressed on NSC in a group of patients with MDS and AML. CD52 is a novel prognostic NSC marker and a potential NSC target in a subset of patients with MDS and AML, which may have clinical implications and may explain clinical effects produced by alemtuzumab in these patients.
Advanced systemic mastocytosis (SM) is a life-threatening neoplasm characterized by uncontrolled growth and accumulation of neoplastic mast cells (MCs) in various organs and a poor survival. So far, ...no curative treatment concept has been developed for these patients. We identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as novel drug target in aggressive SM (ASM) and MC leukemia (MCL). As assessed by immunohistochemistry and PCR, neoplastic MCs expressed substantial amounts of BRD4 in ASM and MCL. The human MCL lines HMC-1 and ROSA also expressed BRD4, and their proliferation was blocked by a BRD4-specific short hairpin RNA. Correspondingly, the BRD4-targeting drug JQ1 induced dose-dependent growth inhibition and apoptosis in HMC-1 and ROSA cells, regardless of the presence or absence of KIT D816V. In addition, JQ1 suppressed the proliferation of primary neoplastic MCs obtained from patients with ASM or MCL (IC50: 100-500 nm). In drug combination experiments, midostaurin (PKC412) and all-trans retinoic acid were found to cooperate with JQ1 in producing synergistic effects on survival in HMC-1 and ROSA cells. Taken together, we have identified BRD4 as a promising drug target in advanced SM. Whether JQ1 or other BET-bromodomain inhibitors are effective in vivo in patients with advanced SM remains to be elucidated.
•Bosutinib synergizes with dasatinib in killing BCR-ABL1T315I+ CML cells.•Synergism was confirmed in primary drug-resistant CML cells carrying BCR-ABL1T315I.•The drug combination produced cooperative ...effects on CD34+/CD38− CML stem cells.•“Bosutinib + dasatinib” may be an interesting approach in TKI-resistant CML.•Forthcoming studies need to clarify the value of the drug combination in vivo.
In chronic myeloid leukemia (CML), resistance against second-generation tyrosine kinase inhibitors (TKI) remains a serious clinical challenge, especially in the context of multi-resistant BCR-ABL1 mutants, such as T315I. Treatment with ponatinib may suppress most of these mutants, including T315I, but is also associated with a high risk of clinically relevant side effects. We screened for alternative treatment options employing available tyrosine kinase inhibitors (TKI) in combination. Dasatinib and bosutinib are two second-generation TKI that bind to different, albeit partially overlapping, spectra of kinase targets in CML cells. This observation prompted us to explore anti-leukemic effects of the combination dasatinib + bosutinib in highly resistant primary CML cells, various CML cell lines (K562, K562R, KU812, KCL22) and Ba/F3 cells harboring various BCR-ABL1 mutant-forms. We found that bosutinib synergizes with dasatinib in inducing growth inhibition and apoptosis in all CML cell lines and in Ba/F3 cells exhibiting BCR-ABL1T315I. Clear synergistic effects were also observed in primary CML cells in all patients tested (n = 20), including drug-resistant cells carrying BCR-ABL1T315I. Moreover, the drug combination produced cooperative or even synergistic apoptosis-inducing effects on CD34+/CD38– CML stem cells. Finally, we found that the drug combination is a potent approach to block the activity of major additional CML targets, including LYN, KIT and PDGFRα. Together, bosutinib and dasatinib synergize in producing anti-leukemic effects in drug-resistant CML cells. Whether such cooperative TKI effects also occur in vivo in patients with drug-resistant CML, remains to be determined in forthcoming studies.
Summary Cancer stem cells, also known as leukemic stem cells (LSC) in the context of leukemias, are an emerging topic in translational oncology and hematology. The Ludwig Boltzmann Institute for ...Hematology and Oncology (LBI HO) was established in 2008 with the aim to translate LSC concepts into clinical practice. Major specific aims of the LBI HO are to identify LSC in various blood cell disorders and to improve anti-leukemic therapies by establishing LSC-targeting and LSC-eradicating approaches with the ultimate aim to translate these concepts into clinical practice. In addition, the LBI HO identified a number of diagnostic and prognostic LSC markers in various blood cell malignancies. Members of the LBI HO have also developed precision medicine tools and personalized medicine approaches around LSC in applied hematology. As a result, diagnosis, prognostication and therapy have improved in the past 10 years. Major disease models are myeloid leukemias and mast cell neoplasms. Finally, the LBI HO consortium launched several projects in the field of open innovation in science where patient-derived initiatives and their input supported the scientific community. Key aims for the future of the LBI HO are to develop LSC-related concepts and strategies further, with the long-term vision to cure more patients with hematologic malignancies.
Systemic mastocytosis (SM) is a hematopoietic neoplasm with a complex pathology and a variable clinical course. Clinical symptoms result from organ infiltration by mast cells (MC) and the effects of ...pro-inflammatory mediators released during MC activation. In SM, growth and survival of MC are triggered by various oncogenic mutant-forms of the tyrosine kinase KIT. The most prevalent variant, D816V, confers resistance against various KIT-targeting drugs, including imatinib. We examined the effects of two novel promising KIT D816V-targeting drugs, avapritinib and nintedanib, on growth, survival, and activation of neoplastic MC and compared their activity profiles with that of midostaurin. Avapritinib was found to suppress growth of HMC-1.1 cells (
V560G) and HMC-1.2 cells (
V560G +
D816V) with comparable IC
values (0.1-0.25 µM). In addition, avapritinib was found to inhibit the proliferation of ROSA
cells, (IC
: 0.1-0.25 µM), ROSA
cells (IC
: 1-5 µM), and ROSA
cells (IC
: 0.1-0.25 µM). Nintedanib exerted even stronger growth-inhibitory effects in these cells (IC
in HMC-1.1: 0.001-0.01 µM; HMC-1.2: 0.25-0.5 µM; ROSA
: 0.01-0.1 µM; ROSA
: 0.5-1 µM; ROSA
: 0.01-0.1 µM). Avapritinib and nintedanib also suppressed the growth of primary neoplastic cells in most patients with SM examined (avapritinib IC
: 0.5-5 µM; nintedanib IC
: 0.1-5 µM). Growth-inhibitory effects of avapritinib and nintedanib were accompanied by signs of apoptosis and decreased surface expression of the transferrin receptor CD71 in neoplastic MC. Finally, we were able to show that avapritinib counteracts IgE-dependent histamine secretion in basophils and MC in patients with SM. These effects of avapritinib may explain the rapid clinical improvement seen during treatment with this KIT inhibitor in patients with SM. In conclusion, avapritinib and nintedanib are new potent inhibitors of growth and survival of neoplastic MC expressing various KIT mutant forms, including D816V, V560G, and K509I, which favors the clinical development and application of these new drugs in advanced SM. Avapritinib is of particular interest as it also blocks mediator secretion in neoplastic MC.
In an attempt to identify novel cell surface markers and targets in leukemic stem cells (LSC) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples ...of patients with AML (n=257), CML (n=134), and controls (n=256: other BM neoplasms, n=116; normal/reactive BM, n=29; idiopathic cytopenia, n=31; lymphoma patients without BM involvement, n=80) for expression of cell surface markers and targets on CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells (PC) by multi-color flow cytometry. In addition, we examined mRNA expression profiles in highly purified CD34+/CD38− stem cells and CD34+/CD38+ PC by gene array- and qPCR analyses. Aberrant LSC expression profiles were identified in all patients examined. In patients with CML, CD34+/CD38− LSC expressed an almost invariable aberration profile defined as CD25+/CD26+/CD56+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38− cells variably displayed aberrant surface antigens, including CD25 (55%), CD96 (35%), CD371 (CLL-1) (75%), and IL-1RAP (60%). With the exception of a subset of FLT3 ITD+ patients (45% of all FLT3-mutated cases), AML LSC did not exhibit CD26. All other markers and targets identified on AML and/or CML LSC, including CD9, CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, neural proliferation and differentiation control-1 antigen (NPDC1), and roundabout-4 (ROBO4), were also detectable on normal hematopoietic stem cells (HSC). However, several surface-targets, including CD33 and CD52, were expressed at higher levels on CD34+/CD38− LSC compared to normal HSC, and antibody-mediated targeting resulted in their selective depletion and in a significantly reduced engraftment of LSC in NSG mice. Since certain surface antigens, like CD47 (IAP), CD243 (MDR1), and CD274 (PD-L1) may contribute to intrinsic drug resistance of LSC, we also examined the expression of these antigens in AML and CML LSC. Regardless of the type of leukemia, LSC were found to express CD47 in all patients examined. MDR-1 was found to be expressed on LSC in 11/39 patients with AML (28%) and 2/22 patients with CML (9%). PD-L1 was not detectable on LSC in untreated BM samples. Exposure of LSC to interferon-gamma (IFN-G, 100 U/ml, 48 hours) resulted in expression of PD-L1 on LSC in all patients with Ph+ CML (control: 100%; IFN-G: 183±40%, p<0.05). Unexpectedly, however, IFN-G was found to induce PD-L1 expression in only 1 out of 11 AML patients tested. IFN-G showed no effects on expression of CD47 or MDR-1 in LSC. We next examined the potential mechanisms of IFN-G-mediated expression of PD-L1 on LSC and screened for drugs capable of counteracting the expression of this resistance-inducing checkpoint molecule. Of all targeted drugs examined (n=10) only the BRD4/MYC inhibitor JQ1 was identified as a regulator of IFN-G-induced PD-L1 expression on CML LSC. In particular, JQ1 (2.5 µM, 48 hours) was found to suppress IFN-G-induced (100 U/ml, 48 hours) upregulation of PD-L1 in primary CML LSC in all donors tested (IFN-G: 183±40% versus IFN-G+JQ1: 136±27% of control) suggesting that checkpoint expression in LSC is regulated by epigenetic mechanisms and MYC activity. Together, we have established cell surface marker- and target expression profiles in CD34+/CD38− AML LSC and CML LSC which should facilitate their enrichment and may support the development of LSC-eradicating treatment concepts. In addition, the unique expression profiles of LSC should provide a powerful basis for diagnostic LSC phenotyping in patients with CML and AML.
Hoermann:Novartis: Honoraria; Gilead: Research Funding; Ariad: Honoraria; Amgen: Honoraria. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Valent:Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria.
Ponatinib is a tyrosine kinase inhibitor (TKI) directed against BCR-ABL1 which is successfully used in patients with
BCR-ABL1
T315I
+ chronic myeloid leukemia (CML). However,
BCR-ABL1
compound ...mutations may develop during therapy in these patients and may lead to drug resistance. Asciminib is a novel drug capable of targeting most BCR-ABL1 mutant-forms, including BCR-ABL1
T315I
, but remains ineffective against most BCR-ABL1
T315I
+ compound mutation-bearing sub-clones. We demonstrate that asciminib synergizes with ponatinib in inducing growth-arrest and apoptosis in patient-derived CML cell lines and murine Ba/F3 cells harboring
BCR-ABL1
T315I
or T315I-including compound mutations. Asciminib and ponatinib also produced cooperative effects on CRKL phosphorylation in BCR-ABL1-transformed cells. The growth-inhibitory effects of the drug combination ‘asciminib+ponatinib’ was further enhanced by hydroxyurea (HU), a drug which has lately been described to suppresses the proliferation of
BCR-ABL1
T315I
+ CML cells. Cooperative drug effects were also observed in patient-derived CML cells. Most importantly, we were able to show that the combinations ‘asciminib+ponatinib’ and ‘asciminib+ponatinib+HU’ produce synergistic apoptosis-inducing effects in CD34
+
/CD38
-
CML stem cells obtained from patients with chronic phase CML or
BCR-ABL1
T315I
+ CML blast phase. Together, asciminib, ponatinib and HU synergize in producing anti-leukemic effects in multi-resistant CML cells, including cells harboring T315I+
BCR-ABL1
compound mutations and CML stem cells. The clinical efficacy of this TKI combination needs to be evaluated within the frame of upcoming clinical trials.
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