Kelly Mitchell

New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Direct Activation of BAX by BTSA1 Overcomes Apoptosis Resistance in Acute Myeloid Leukemia

The BCL-2 family protein BAX is a central mediator of apoptosis. Overexpression of anti-apoptotic BCL-2 proteins contributes to tumor development and resistance to therapy by suppressing BAX and its activators. We report the discovery of BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site and induces conformational changes to BAX leading to BAX-mediated apoptosis. BTSA1-induced BAX activation effectively promotes apoptosis in leukemia cell lines and patient samples while sparing healthy cells. BAX expression levels and cytosolic conformation regulate sensitivity to BTSA1. BTSA1 potently suppressed human acute myeloid leukemia (AML) xenografts and increased host survival without toxicity. This study provides proof-of-concept for direct BAX activation as a treatment strategy in AML.

Pharmacological inhibition of the transcription factor PU.1 in leukemia

The transcription factor PU.1 is often impaired in patients with acute myeloid leukemia (AML). Here, we used AML cells that already had low PU.1 levels and further inhibited PU.1 using either RNA interference or, to our knowledge, first-in-class small-molecule inhibitors of PU.1 that we developed specifically to allosterically interfere with PU.1-chromatin binding through interaction with the DNA minor groove that flanks PU.1-binding motifs. These small molecules of the heterocyclic diamidine family disrupted the interaction of PU.1 with target gene promoters and led to downregulation of canonical PU.1 transcriptional targets. shRNA or small-molecule inhibition of PU.1 in AML cells from either PU.1lo mutant mice or human patients with AML-inhibited cell growth and clonogenicity and induced apoptosis. In murine and human AML (xeno)transplantation models, treatment with our PU.1 inhibitors decreased tumor burden and resulted in increased survival. Thus, our study provides proof of concept that PU.1 inhibition has potential as a therapeutic strategy for the treatment of AML and for the development of small-molecule inhibitors of PU.1.

Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia

Dual inhibition of MDMX and MDM2 by an α-helical p53-stapled peptide (ALRN-6924) results in robust antitumor activity in acute myeloid leukemia. A new staple of leukemia treatment? As suggested by their name, tumor suppressor genes prevent tumorigenesis, and their expression or activity is often lost in cancer cells. One of the best known tumor suppressors is p53, which is inactivated in a variety of cancer types, often through up-regulation of its endogenous suppressors. Despite numerous attempts to reactivate p53 by a variety of approaches, none have successfully advanced beyond clinical trials thus far. Now, Carvajal et al. applied yet another tactic to restore p53 activity by using a stapled peptide to inactivate both of its endogenous inhibitors, for situations where the tumor suppressor is inactive but not completely lost. The authors demonstrated the effectiveness of this approach in human acute myeloid leukemia using in vitro and in vivo models, along with preliminary testing in a patient with leukemia. The tumor suppressor p53 is often inactivated via its interaction with endogenous inhibitors mouse double minute 4 homolog (MDM4 or MDMX) or mouse double minute 2 homolog (MDM2), which are frequently overexpressed in patients with acute myeloid leukemia (AML) and other cancers. Pharmacological disruption of both of these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in cancers with wild-type p53. Selective targeting of this pathway has thus far been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. We demonstrate that dual MDMX/MDM2 inhibition with a stapled α-helical peptide (ALRN-6924), which has recently entered phase I clinical testing, produces marked antileukemic effects. ALRN-6924 robustly activates p53-dependent transcription at the single-cell and single-molecule levels and exhibits biochemical and molecular biological on-target activity in leukemia cells in vitro and in vivo. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibits cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patient cells, including leukemic stem cell–enriched populations, and disrupts functional clonogenic and serial replating capacity. Furthermore, ALRN-6924 markedly improves survival in AML xenograft models. Our study provides mechanistic insight to support further testing of ALRN-6924 as a therapeutic approach in AML and other cancers with wild-type p53.

Abstract C225: IL1RAP as functionally relevant target for stem-cell directed therapy in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS).

Despite the established use of poly-chemotherapy, relapse continues to be the most common cause of death in AML and MDS and cure rates remain below 20%. AML/MDS arise following the accumulation of stepwise genetic and epigenetic changes in hematopoietic stem and progenitor cells (HSPC). Utilizing a novel strategy of parallel transcriptional analysis of sorted HSPC populations in distinct subtypes of AML, we compared the gene expression in AML HSPC with identical compartments from age-matched healthy controls and identified Interleukin 1 receptor accessory protein (IL1RAP) as one of the most significantly upregulated genes in HSPC in all examined subtypes of AML. Fluorescence in situ hybridization of sorted IL1RAP+ and IL1RAP- cells from patients with monosomy 7 AML (-7) indicated that the aberrant clone was restricted to IL1RAP+ cells, demonstrating that IL1RAP overexpression is a distinguishing feature of the -7 clone. Multivariate analysis of a large cohort of patients with normal karyotype AML showed that patients with high IL1RAP levels had inferior overall survival than patients with lower IL1RAP levels, suggesting an independent prognostic value for this molecule in AML. IL1RAP expression levels in MDS were found elevated on stem cells of patients with high risk disease, proposing a role of IL1RAP in higher risk MDS and progression to AML. Downregulation of IL1RAP expression by lentivirally expressed shRNAs decreased clonogenicity in cell lines and AML/MDS primary patient samples, induced apoptosis of AML cells, and reduced proliferation of AML cells and infiltration of hematopoietic organs in vivo. IL1RAP is a transmembrane protein required for signaling through several receptors of the IL1 family. Downregulation of IL1RAP expression in AML cells led to phosphorylation changes in several kinases and their substrates suggesting participation of IL1RAP in multiple signaling pathways and highlighting its potential as therapeutic target. We investigated whether inhibition of IL1RAP with pharmacological compounds is feasible and effective. Antibody-mediated inhibition of IL1RAP led to inhibition of AML cell growth in vitro. In addition, we designed peptides to interfere with IL1RAP-receptor interactions which lead to inhibition of AML cell growth. Both types of agents are being further tested and optimized. In summary, our study reveals IL1RAP as aberrantly expressed on HSPC of AML and high-risk MDS patients. Inhibition of IL1RAP is feasible and functionally effective, and thus has the potential to lead to novel therapies specifically directed at such stem cells. Beyond IL1RAP, our study provides a map of dysregulated transcripts in HSPC from patients with AML, which may offer further opportunities for therapeutic intervention. The strategy of comparative analysis of sorted stem and progenitor cells in cancer versus healthy controls may be applicable to other type of cancers with a suspected stem cell origin, and instrumental for the identification of targets for stem cell-directed therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C225. Citation Format: Laura Barreyro, Kelly Mitchell, Britta Will, Boris Bartholdy, Li Zhou, Tihomira Todorova, Robert Stanley, Susana Ben-Neriah, Cristina Montagna, Samir Parekh, Andrea Pellagatti, Jacqueline Boultwood, Elisabeth Paietta, Rhett Ketterling, Larry Cripe, Hugo Fernandez, Peter Greenberg, Jacob Rowe, Martin Tallman, Christian Steidl, Constantine Mitsiades, Amit Verma, Ulrich Steidl. IL1RAP as functionally relevant target for stem-cell directed therapy in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C225.

IL1RAP potentiates multiple oncogenic signaling pathways in AML

The surface molecule interleukin-1 receptor accessory protein (IL1RAP) is consistently overexpressed across multiple genetic subtypes of acute myeloid leukemia (AML) and other myeloid malignancies, including at the stem cell level, and is emerging as a novel therapeutic target. However, the cell-intrinsic functions of IL1RAP in AML cells are largely unknown. Here, we show that targeting of IL1RAP via RNA interference, genetic deletion, or antibodies inhibits AML pathogenesis in vitro and in vivo, without perturbing healthy hematopoietic function or viability. Furthermore, we found that the role of IL1RAP is not restricted to the IL-1 receptor pathway, but that IL1RAP physically interacts with and mediates signaling and pro-proliferative effects through FLT3 and c-KIT, two receptor tyrosine kinases with known key roles in AML pathogenesis. Our study provides a new mechanistic basis for the efficacy of IL1RAP targeting in AML and reveals a novel role for this protein in the pathogenesis of the disease.

A myeloid tumor suppressor role for NOL3

Despite the identification of several oncogenic driver mutations leading to constitutive JAK–STAT activation, the cellular and molecular biology of myeloproliferative neoplasms (MPN) remains incompletely understood. Recent discoveries have identified underlying disease-modifying molecular aberrations contributing to disease initiation and progression. Here, we report that deletion of Nol3 (Nucleolar protein 3) in mice leads to an MPN resembling primary myelofibrosis (PMF). Nol3−/− MPN mice harbor an expanded Thy1+LSK stem cell population exhibiting increased cell cycling and a myelomonocytic differentiation bias. Molecularly, this phenotype is mediated by Nol3−/−-induced JAK–STAT activation and downstream activation of cyclin-dependent kinase 6 (Cdk6) and Myc. Nol3−/− MPN Thy1+LSK cells share significant molecular similarities with primary CD34+ cells from PMF patients. NOL3 levels are decreased in CD34+ cells from PMF patients, and the NOL3 locus is deleted in a subset of patients with myeloid malignancies. Our results reveal a novel genetic PMF-like mouse model and identify a tumor suppressor role for NOL3 in the pathogenesis of myeloid malignancies.

Abstract C38: Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. AML is hallmarked by a differentiation block and patient outcomes remain poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. Hence the development of novel targeted therapies for treatment of AML subtypes are required. Of note, inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients9 cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Treatment of primary IDH1 mutant AML patients9 cells ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in leukemic blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including “Cell Cycle”, “G1/S transition”, “Cellular growth and proliferation”, and “Cell death and survival”. Taken together, our findings provide novel insight into the effects of inhibition of mutant IDH1 in primary AML patients9 cells and open avenues for future investigations with these and other novel allosteric inhibitors for targeting IDH1 mutants in leukemia and possibly in other cancers. Citation Format: Ujunwa C. Okoye-Okafor, Boris Bartholdy, Jessy Cartier, Enoch Gao, Beth Pietrak, Alan R. Rendina, Cynthia Rominger, Chad Quinn, Angela Smallwood, Ken Wiggall, Alexander Reif, Stan Schmidt, Hongwei Qi, Huizhen Zhao, Gerard Joberty, Maria Faelth-Savitski, Marcus Bantscheff, Gerard Drewes, Chaya Duraiswami, Pat Brady, Swathi-Rao Narayanagari, Ileana Antony-Debre, Kelly Mitchell, Heng Rui Wang, Yun-Ruei Kao, Maximilian Christopeit, Luis Carvajal, Laura Barreyro, Elisabeth Paietta, Britta Will, Nestor Concha, Nicholas D. Adams, Benjamin Schwartz, Michael T. McCabe, Jaroslav Maciejewski, Amit Verma, Ulrich Steidl. Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C38.