Viktoras Frismantas

The activating STAT5B N642H mutation is a common abnormality in pediatric T-cell acute lymphoblastic leukemia and confers a higher risk of relapse

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes that accounts for approximately 15% of pediatric acute lymphoblastic leukemias. A variety of genetic events affecting cellular processes such as the cell cycle, differentiation and survival have been identified in

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options

TCF3-HLF−positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF−positive and treatment-responsive TCF3-PBX1−positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Cell and Molecular Determinants of In Vivo Efficacy of the BH3 Mimetic ABT-263 against Pediatric Acute Lymphoblastic Leukemia Xenografts

Purpose: Predictive biomarkers are required to identify patients who may benefit from the use of BH3 mimetics such as ABT-263. This study investigated the efficacy of ABT-263 against a panel of patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts and utilized cell and molecular approaches to identify biomarkers that predict in vivo ABT-263 sensitivity. Experimental Design: The in vivo efficacy of ABT-263 was tested against a panel of 31 patient-derived ALL xenografts composed of MLL-, BCP-, and T-ALL subtypes. Basal gene expression profiles of ALL xenografts were analyzed and confirmed by quantitative RT-PCR, protein expression and BH3 profiling. An in vitro coculture assay with immortalized human mesenchymal cells was utilized to build a predictive model of in vivo ABT-263 sensitivity. Results: ABT-263 demonstrated impressive activity against pediatric ALL xenografts, with 19 of 31 achieving objective responses. Among BCL2 family members, in vivo ABT-263 sensitivity correlated best with low MCL1 mRNA expression levels. BH3 profiling revealed that resistance to ABT-263 correlated with mitochondrial priming by NOXA peptide, suggesting a functional role for MCL1 protein. Using an in vitro coculture assay, a predictive model of in vivo ABT-263 sensitivity was built. Testing this model against 11 xenografts predicted in vivo ABT-263 responses with high sensitivity (50%) and specificity (100%). Conclusion: These results highlight the in vivo efficacy of ABT-263 against a broad range of pediatric ALL subtypes and shows that a combination of in vitro functional assays can be used to predict its in vivo efficacy. Clin Cancer Res; 20(17); 4520–31. ©2014 AACR.

Ex vivo drug response profiling detects recurrent sensitivity patterns in drug-resistant acute lymphoblastic leukemia

Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2-inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.

Targeting BET proteins improves the therapeutic efficacy of BCL-2 inhibition in T-cell acute lymphoblastic leukemia.

Inhibition of anti-apoptotic BCL-2 (B-cell lymphoma 2) has recently emerged as a promising new therapeutic strategy for the treatment of a variety of human cancers, including leukemia. Here, we used T-cell acute lymphoblastic leukemia (T-ALL) as a model system to identify novel synergistic drug combinations with the BH3 mimetic venetoclax (ABT-199). In vitro drug screening in primary leukemia specimens that were derived from patients with high risk of relapse or relapse and cell lines revealed synergistic activity between venetoclax and the BET (bromodomain and extraterminal) bromodomain inhibitor JQ1. Notably, this drug synergism was confirmed in vivo using T-ALL cell line and patient-derived xenograft models. Moreover, the therapeutic benefit of this drug combination might, at least in part, be mediated by an acute induction of the pro-apoptotic factor BCL2L11 and concomitant reduction of BCL-2 upon BET bromodomain inhibition, ultimately resulting in an enhanced binding of BIM (encoded by BCL2L11) to BCL-2. Altogether, our work provides a rationale to develop a new type of targeted combination therapy for selected subgroups of high-risk leukemia patients.

PHARMACOLOGICAL ACTIVITY OF CB-103 IN HAEMATOLOGICAL MALIGNANCIES – AN ORAL PAN-NOTCH INHIBITOR WITH A NOVEL MODE OF ACTION

Introduction: NOTCH signalling is critical during embryonic development as well as for the regulation of self‐renewing tissues. Aberrant activation of NOTCH signalling in cancer leads to sustained proliferation, escape from apoptosis, loss of differentiation capacity, increased invasion and metastasis and is a negative prognostic factor. Overactivation of the NOTCH pathway due to various genetic lesions (over expressions, mutations, translocations), is a major driver for NOTCH‐dependent cancers and resistance to standard of care treatment.Several therapeutic NOTCH inhibitors are currently in clinical testing with a) monoclonal antibodies (mAbs) against NOTCH ligands and receptors and b) gamma‐secretase inhibitors (GSIs). However clinical activity and exposure of these in clinical studies were limited due to gastro‐intestinal toxicities. In haematological malignancies with constitutive NOTCH activation (gene fusion due to chromosomal translocations or NOTCH mutations), mAbs and GSIs will have very limited clinical benefits. Here we report, discovery and development of a novel orally active small molecule inhibitor (CB‐103) of the NOTCH pathway. CB‐103 blocks NOTCH signalling by a novel mode of action, directly targeting the NOTCH transcriptional activation complex. We will further present the in vitro and in vivo pharmacological characterization of CB‐103. Methods: Primary pharmacodynamic (PD) studies were conducted to investigate CB‐103 in relation to its desired therapeutic effect for treating advanced haematological malignancies as a NOTCH pathway inhibitor. Regarding the PD effect, in vitro studies demonstrated for CB‐103 a dose‐dependent decrease in NOTCH signalling activation with a unique mechanism compared to GSIs and mAbs. The NOTCH inhibitory potential of CB‐103 was further confirmed by downregulation of NOTCH target genes in human T‐cell acute lymphoblastic leukaemia (T‐ALL), suggesting therapeutic efficacy of CB‐103 in the context of T‐ALL. In a panel of >120 cell lines of various malignancies CB‐103 was active on a subset of 24 cancer cell lines, including different lymphomas. Moreover, CB‐103 demonstrated anti‐NOTCH activity in the Triple‐Negative Breast Cancer (TNBC) HCC1187 cell line, being resistant to GSIs due to a NOTCH2 chromosomal translocation. Results: We demonstrate that in vitro CB‐103 potently inhibits NOTCH signalling in various lymphoma and leukaemia cell lines, and T‐ALL blasts derived from relapse/refractory patients. In addition, CB‐103 exhibited anti‐tumor efficacy in in vivo models of NOTCH‐ driven T‐ALL using T‐ALL cell lines and PDx models. Conclusions: Toxicology studies have revealed an excellent safety profile in the expected human therapeutic dose range. Clinical development of CB‐103 with a first‐in‐human Phase I/IIA clinical study in advanced HL, selected NHL indications and solid tumours is under preparation.

Abstract 338: A novel small molecule inhibitor of the Notch transcription activation complex

NOTCH signaling is a developmental pathway known to play critical roles during embryonic development as well as for the regulation of self-renewing tissues. Aberrant activation of NOTCH signaling leads to deregulation of the self-renewal process resulting in sustained proliferation, evasion of cell death, loss of differentiation capacity, invasion and metastasis, all of which are hallmarks of cancer. Over activation of NOTCH in human cancers can be a consequence of over expression of NOTCH ligands/receptors, GOF mutations in NOTCH receptors as well as chromosomal translocations leading to constitutive activation of the pathway. Given the importance of Notch signaling in human cancers, several therapeutic approaches have been utilized to block NOTCH signaling. Two of these strategies are; a) the use of monoclonal blocking antibodies (Mabs) against NOTCH ligands and receptors and b) the use of small molecule γ-secretase inhibitors (GSIs). However, these approaches can only be effective if tumor cells express full-length ligand or receptor molecules. On the contrary, in human cancers harbouring NOTCH gene fusion due to chromosomal translocations, the use of Mabs and GSIs will have very limited clinical benefits. A third, yet not fully explored approach could be the blockage of NOTCH signalling by targeting the most downstream event in the NOTCH cascade i,e NOTCH transcriptional activation complex using small molecule inhibitors. Here we report discovery and identification of a novel, orally-active small molecule inhibitor, named CB-103, of the NOTCH pathway that blocks NOTCH signaling by targeting the NOTCH transcriptional activation complex in the nucleus. CB-103 has shown the ability to block NOTCH signalling in human cancer cell lines, induce neurogenic phenotype in drosophila, induce muscle cell differentiation and inhibit NOTCH dependent cellular processes in mice. In addition, CB-103 exhibit anti-tumor efficacy in a xenograft model of human triple negative breast cancer resistant to GSIs and Mabs against NOTCH ligands/receptors. Furthermore, CB-103 has shown a remarkable activity in PDX models of human T-ALL harbouring activation of the NOTCH pathway. Additional studies are underway on several analogs of CB-103 to determine ADME/PK/PD profile and nominate a development candidate for further clinical development of this novel inhibitor of the NOTCH pathway. Citation Format: Rajwinder Lehal, Viktoras Frismantas, Sylvain Loubery, Viktoria Reinmuller, Gerardo Turcatti, Marcos Gonzalez-Gaitan, Jean-Pierre Bourquin, Freddy Radtke. A novel small molecule inhibitor of the Notch transcription activation complex. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 338.

Abstract 493: Drug response profiling to inform individualized treatment approaches in high risk leukemia

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Novel treatment approaches are needed for patients with acute lymphoblastic leukemia (ALL) who respond poorly to current therapies. The genotypic diversity identified recently by next generation sequencing technologies within ALL calls for individualized novel strategies. However, functional correlations of oncogenic lesions with drug response profiles are ill defined for ALL. We have established an imaging-based cell viability analysis platform to generate drug response profiles for primary patient-derived ALL samples co-cultured with mesenchymal stroma cells, expecting to derive functional information directly from individual patient samples. Such response profiles may mirror perturbations in relevant cellular programs that could be exploited therapeutically. Our pipeline integrates high-content screening, newly developed bioinformatics tools and biochemical approaches. We screened a library of 65 compounds for activity in 37 precursor B-ALL and 23 T-ALL samples including refractory cases. Cross-sample comparisons revealed that cells from relapsed refractory patients showed a more resistant phenotype for most of the drugs. While only a few agents including genotoxic drugs showed activity across all samples, we detected selective activity of given drugs that distinguish patient sample groups. MLL-rearranged and TCF3-HLF-positive ALL samples as well as a subgroup of T-ALL cases were highly sensitive to the BCL-2 specific BH3-mimetic ABT-199 suggesting BCL-2 dependency for these cases. Multiparametric analyses of in vitro responses predicted in vivo activity of ABT-199 in xenografted mice. Moreover, we could identify synergistic activity of ABT-199 with clinical and preclinical compounds, such as topotecan or epigenetic modifiers. Our drug response profiling pipeline contributes to the identification of distinct vulnerabilities in leukemia and may facilitate the selection of candidate drugs for further development into clinical application. Citation Format: Viktoras Frismantas, Anna Rinaldi, Maria Pamela Dobay, Salome Higi, Sabrina Eugster, Blerim Marovca, Peter Horvath, Mauzro Delorenzi, Joachim Kunz, Obul R. Bandapalli, Gunnar Cario, Martin Stanulla, Andreas E. Kulozik, Martina Muckenthaler, Cornelia Eckert, Thomas Radimerski, Jean-Pierre Bourquin, Beat C. Bornhauser. Drug response profiling to inform individualized treatment approaches in high risk leukemia. [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 493. doi:10.1158/1538-7445.AM2015-493