Tim Sauer

Allogeneic Transplantation Versus Chemotherapy as Postremission Therapy for Acute Myeloid Leukemia: A Prospective Matched Pairs Analysis

PURPOSE The majority of patients with acute myeloid leukemia (AML) who achieve complete remission (CR) relapse with conventional postremission chemotherapy. Allogeneic stem-cell transplantation (alloSCT) might improve survival at the expense of increased toxicity. It remains unknown for which patients alloSCT is preferable. PATIENTS AND METHODS We compared the outcome of 185 matched pairs of a large multicenter clinical trial (AMLCG99). Patients younger than 60 years who underwent alloSCT in first remission (CR1) were matched to patients who received conventional postremission therapy. The main matching criteria were AML type, cytogenetic risk group, patient age, and time in first CR. RESULTS In the overall pairwise compared AML population, the projected 7-year overall survival (OS) rate was 58% for the alloSCT and 46% for the conventional postremission treatment group (P = .037; log-rank test). Relapse-free survival (RFS) was 52% in the alloSCT group compared with 33% in the control group (P < .001). OS was significantly better for alloSCT in patient subgroups with nonfavorable chromosomal aberrations, patients older than 45 years, and patients with secondary AML or high-risk myelodysplastic syndrome. For the entire patient cohort, postremission therapy was an independent factor for OS (hazard ratio, 0.66; 95% CI, 0.49 to 0.89 for alloSCT v conventional chemotherapy), among age, cytogenetics, and bone marrow blasts after the first induction cycle. CONCLUSION AlloSCT is the most potent postremission therapy for AML and is particularly active for patients 45 to 59 years of age and/or those with high-risk cytogenetics.

Loss of the histone methyltransferase EZH2 induces resistance to multiple drugs in acute myeloid leukemia

In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.

Prognostic Impact of Bcl-2 Depends on Tumor Histology and Expression of MALAT-1 lncRNA in Non–Small-Cell Lung Cancer

Introduction: Apoptosis is a crucial pathway in tumor growth and metastatic development. Apoptotic proteins regulate the underlying molecular cascades and are thought to modulate the tumor response to chemotherapy and radiation. However, the prognostic value of the expression of apoptosis regulators in localized non–small-cell lung cancer (NSCLC) is still unclear. Methods: We investigated the protein expression of apoptosis regulators Bcl-2, Bcl-xl, Mcl-1, and pp32/PHAPI, and the expression of the lncRNA MALAT-1 in tumor samples from 383 NSCLC patients (median age: 65.6 years; 77.5% male; paraffin-embedded tissue microarrays). For statistical analysis correlation tests, Log rank tests and Cox proportional hazard models were applied. Results: Tumor histology was significantly associated with the expression of Bcl-2, Bcl-xl and Mcl-1 (all p < 0.001). Among the tested apoptotic markers only Bcl-2 demonstrated prognostic impact (hazard ratio = 0.64, p = 0.012). For NSCLC patients with non-adenocarcinoma histology, Bcl-2 expression was associated with increased overall survival (p = 0.036). Besides tumor histology, prognostic impact of Bcl-2 was also found to depend on MALAT-1 lncRNA expression. Gene expression analysis of A549 adenocarcinoma cells with differential MALAT-1 lncRNA expression demonstrated an influence on the expression of Bcl-2 and its interacting proteins. Conclusions: Bcl-2 expression was specifically associated with superior prognosis in localized NSCLC. An interaction of Bcl-2 with MALAT-1 lncRNA expression was revealed, which merits further investigation for risk prediction in resectable NSCLC patients.

Transcription factor CCAAT/enhancer-binding protein alpha and critical circadian clock downstream target gene PER2 are highly deregulated in diffuse large B-cell lymphoma

Abstract Disturbances of circadian rhythms and mammalian clock genes have been implicated in the etiologies of many chronic illnesses, including cancer. We show that transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha)-regulated PER2 activation is a potential tumor suppressor pathway in diffuse large B-cell lymphoma (DLBCL), one of the commonest types of mature B-cell lymphoma. Expression analysis of human B-cell lymphoma samples including DLBCL (n = 50), mantle cell (n = 21), follicular (n = 25) and Burkitt (n = 18) lymphoma revealed markedly down-regulated CEBPA and PER2 mRNA levels exclusively in DLBCL samples compared to control lymphatic tissue. We demonstrated direct regulation of the circadian core clock gene PER2 by C/EBPalpha in the pro-B cell line Ba/F3, and forced expression of PER2 resulted in decreased proliferation, G0/G1 cell cycle arrest and increased rates of apoptosis. Interestingly, treatment of human DLBCL cell lines with the histone deacetylase-inhibitor suberoylanilide hydroxamic acid (SAHA) significantly increased the expression of C/EBPalpha and Per2, accompanied by cell growth inhibition; in contrast, siRNA knockdown of CEBPA reduced the anti-proliferative effect of SAHA treatment. Our results show for the first time that C/EBPalpha with its associated direct core clock gene target, PER2, are highly deregulated in DLBCL, suggesting an important tumor suppressive pathway in the pathogenesis of this lymphoma entity.

A Phase I Dose Escalation Study of the Triple Angiokinase Inhibitor Nintedanib Combined with Low-Dose Cytarabine in Elderly Patients with Acute Myeloid Leukemia

Nintedanib (BIBF 1120), a potent multikinase inhibitor of VEGFR-1/-2/-3, FGFR-1/-2/-3 and PDGFR-α/-β, exerts growth inhibitory and pro-apoptotic effects in myeloid leukemic cells, especially when used in combination with cytarabine. This phase I study evaluated nintedanib in combination with low-dose cytarabine (LDAC) in elderly patients with untreated or relapsed/refractory acute myeloid leukemia (AML) ineligible for intensive chemotherapy in a 3+3 design. Nintedanib (dose levels 100, 150, and 200 mg orally twice daily) and LDAC (20 mg subcutaneous injection twice daily for 10 days) were administered in 28-day cycles. Dose-limiting toxicity (DLT) was defined as non-hematological severe adverse reaction CTC grade ≥ 4 with possible or definite relationship to nintedanib. Between April 2012 and October 2013, 13 patients (median age 73 [range: 62–86] years) were enrolled. One patient did not receive study medication and was replaced. Nine (69%) patients had relapsed or refractory disease and 6 (46%) patients had unfavorable cytogenetics. The most frequently reported treatment-related adverse events (AE) were gastrointestinal events. Twelve SAEs irrespective of relatedness were reported. Two SUSARs were observed, one fatal hypercalcemia and one fatal gastrointestinal infection. Two patients (17%) with relapsed AML achieved a complete remission (one CR, one CRi) and bone marrow blast reductions without fulfilling PR criteria were observed in 3 patients (25%). One-year overall survival was 33%. Nintedanib combined with LDAC shows an adequate safety profile and survival data are promising in a difficult-to-treat patient population. Continuation of this trial with a phase II recommended dose of 2 x 200 mg nintedanib in a randomized, placebo-controlled phase II study is planned. The trial is registered to EudraCT as 2011-001086-41. Trial Registration: ClinicalTrials.gov NCT01488344

Abstract 4430: Loss of the histone methyltransferase EZH2 induces chemoresistance in acute myeloid leukemia (AML)

Resistance to chemotherapy and subsequent relapse is the most challenging issue in the treatment of patients with Acute Myeloid Leukemia (AML). However, the underlying mechanisms still remain incompletely understood. Here we report that loss of the histone methyltransferase EZH2 and subsequent reduction of H3K27 trimethylation contribute to chemoresistance in AML. In Myelodysplastic Syndrome (MDS) and Myeloproliferative Neoplasms (MPN) EZH2 is often inactivated due to mutations which is associated with poor prognosis. By use of quantitative PCR and immunohistochemistry we show that a decrease of EZH2 mRNA and protein also correlated with a poor prognosis of AML patients indicating a tumor suppressor role of EZH2 in AML. EZH2 is located on chromosome 7q36.1 and it is not yet fully clear whether EZH2 expression is affected in MDS and AML patients with del(7)/del(7q) who are largely refractory to chemotherapy and have a poor prognosis. We found EZH2 levels to be reduced in del(7)/del(7q) AML patients as determined by Western Blot. Notably, the reduction of EZH2 protein levels via treatment with H3K27 methyltransferase inhibitors or lentiviral knockdown was sufficient to induce chemoresistance of Normal Karyotype (NK)- AML blasts and cell lines in vitro and in a xenograft mouse model. Furthermore, we observed that EZH2 loss occurred during the acquisition of drug resistance in a Tyrosine Kinase Inhibitor- and Cytarabine (AraC)- resistant AML cell line. Pharmacological inhibition of CDK1 and treatment with the proteasome inhibitor Bortezomib, respectively, increased EZH2 protein and restored drug sensitivity. Functionally, the loss of EZH2 directly induced upregulation of HOX genes, suggesting a stem-cell-like signature to be associated with the resistance phenotype, which could be reverted by Bortezomib treatment. To evaluate the potential of Bortezomib to affect EZH2 levels in patient blasts we treated primary NK-AML blasts collected at diagnosis ex vivo with Bortezomib. In almost all samples Bortezomib treatment induced cytotoxic effects. In 5 out of 10 patients the EZH2 protein level could be increased by Bortezomib treatment. We furthermore examined the sensitivity of patient samples to AraC, Bortezomib or combined treatment. Notably, for those patients with increased EZH2 levels after Bortezomib exposure we found a significantly decreased cell survival for the combined treatment compared to single-agent treatment. Our data strongly suggest that restoration of EZH2 protein levels e.g. via proteasome inhibitors and thereby restoration of EZH2 function might be a novel promising approach to increase therapy response in AML. Citation Format: Stefanie Gollner, Shuchi Agrawal-Singh, Tino Schenk, Hans-Ulrich Klein, Christian Rohde, Tim Sauer, Mads Lerdrup, Sigal Tavor, Friedrich Stolzel, Gerhard Ehninger, Gabriele Kohler, Martin Dugas, Arthur Zelent, Christian Thiede, Wolfgang E. Berdel, Klaus Hansen, Carsten Muller-Tidow. Loss of the histone methyltransferase EZH2 induces chemoresistance in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4430. doi:10.1158/1538-7445.AM2015-4430