Christina D. Drenberg

The genomic landscape of hypodiploid acute lymphoblastic leukemia

The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole-genome and exome sequencing of 40 cases, identified two subtypes that differ in the severity of aneuploidy, transcriptional profiles and submicroscopic genetic alterations. Near-haploid ALL with 24–31 chromosomes harbor alterations targeting receptor tyrosine kinase signaling and Ras signaling (71%) and the lymphoid transcription factor gene IKZF3 (encoding AIOLOS; 13%). In contrast, low-hypodiploid ALL with 32–39 chromosomes are characterized by alterations in TP53 (91.2%) that are commonly present in nontumor cells, IKZF2 (encoding HELIOS; 53%) and RB1 (41%). Both near-haploid and low-hypodiploid leukemic cells show activation of Ras-signaling and phosphoinositide 3-kinase (PI3K)-signaling pathways and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.

Combined Targeting of JAK2 and Bcl-2/Bcl-xL to Cure Mutant JAK2-Driven Malignancies and Overcome Acquired Resistance to JAK2 Inhibitors

Summary To design rational therapies for JAK2-driven hematological malignancies, we functionally dissected the key survival pathways downstream of hyperactive JAK2. In tumors driven by mutant JAK2, Stat1, Stat3, Stat5, and the Pi3k and Mek/Erk pathways were constitutively active, and gene expression profiling of TEL-JAK2 T-ALL cells revealed the upregulation of prosurvival Bcl-2 family genes. Combining the Bcl-2/Bcl-xL inhibitor ABT-737 with JAK2 inhibitors mediated prolonged disease regressions and cures in mice bearing primary human and mouse JAK2 mutant tumors. Moreover, combined targeting of JAK2 and Bcl-2/Bcl-xL was able to circumvent and overcome acquired resistance to single-agent JAK2 inhibitor treatment. Thus, inhibiting the oncogenic JAK2 signaling network at two nodal points, at the initiating stage (JAK2) and the effector stage (Bcl-2/Bcl-xL), is highly effective and provides a clearly superior therapeutic benefit than targeting just one node. Therefore, we have defined a potentially curative treatment for hematological malignancies expressing constitutively active JAK2.

Panobinostat Enhances Cytarabine and Daunorubicin Sensitivities in AML Cells through Suppressing the Expression of BRCA1, CHK1, and Rad51

Acute myeloid leukemia (AML) remains a challenging disease to treat and urgently requires new therapies to improve its treatment outcome. In this study, we investigated the molecular mechanisms underlying the cooperative antileukemic activities of panobinostat and cytarabine or daunorubicin (DNR) in AML cell lines and diagnostic blast samples in vitro and in vivo. Panobinostat suppressed expression of BRCA1, CHK1, and RAD51 in AML cells in a dose-dependent manner. Further, panobinostat significantly increased cytarabine- or DNR-induced DNA double-strand breaks and apoptosis, and abrogated S and/or G2/M cell cycle checkpoints. Analogous results were obtained by shRNA knockdown of BRCA1, CHK1, or RAD51. Cotreatment of NOD-SCID-IL2Rγnull mice bearing AML xenografts with panobinostat and cytarabine significantly increased survival compared to either cytarabine or panobinostat treatment alone. Additional studies revealed that panobinostat suppressed the expression of BRCA1, CHK1, and RAD51 through downregulation of E2F1 transcription factor. Our results establish a novel mechanism underlying the cooperative antileukemic activities of these drug combinations in which panobinostat suppresses expression of BRCA1, CHK1, and RAD51 to enhance cytarabine and daunorubicin sensitivities in AML cells.

Integrating Clinical Pharmacology Concepts in Individualized Therapy With Tyrosine Kinase Inhibitors

Tyrosine kinases have emerged as important tumor targets for the design of potent and selective inhibitors. Eighteen of these tyrosine kinase inhibitors (TKIs) have already been approved for the treatment of diseases that were previously essentially resistant to standard chemotherapy. Major efforts are ongoing that focus on the development of companion diagnostics for investigational and approved TKIs, as well as on integrating clinical pharmacology principles in clinical practice to decrease toxicity and improve efficacy.

Inherited variation in OATP1B1 is associated with treatment outcome in acute myeloid leukemia

Using broad interrogation of clinically relevant drug absorption, distribution, metabolism, and excretion (ADME) genes on the DMET platform, we identified a genetic variant in SLCO1B1 (rs2291075; c.597C>T), encoding the transporter OATP1B1, associated with event‐free (P = 0.006, hazard ratio = 1.74) and overall survival (P = 0.012, hazard ratio = 1.85) in children with de novo acute myeloid leukemia (AML). Lack of SLCO1B1 expression in leukemic blasts suggested the association might be due to an inherited rather than a somatic effect. rs2291075 was in strong linkage with known functional variants rs2306283 (c.388A>G) and rs4149056 (c.521T>C). Functional studies in vitro determined that four AML‐directed chemotherapeutics (cytarabine, daunorubicin, etoposide, and mitoxantrone) are substrates for OATP1B1 and the mouse ortholog Oatp1b2. In vivo pharmacokinetic studies using Oatp1b2‐deficient mice further confirmed our results. Collectively, these findings demonstrate an important role for OATP1B1 in the systemic pharmacokinetics of multiple drugs used in the treatment of AML and suggest that inherited variability in host transporter function influences the effectiveness of therapy.

Transcriptome profiling of patient derived xenograft models established from pediatric acute myeloid leukemia patients confirm maintenance of FLT3-ITD mutation.

Christina D. Drenberg, Daelynn R. Buelow, Stanley B. Pounds, Yong-Dong Wang, David Finkelstein, Richard J. Rahija, Sheila A. Shurtleff, Jeffrey E. Rubnitz, Hiroto Inaba, Tanja A. Gruber, Jeffery M. Klco and Sharyn D. Baker Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA; Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA; Department of Biostatics, St. Jude Children’s Research Hospital, Memphis, TN, USA; Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA; Animal Resource Center, St. Jude Children’s Research Hospital, Memphis, TN, USA; Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA; Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN, USA

ABCC4 Is a Determinant of Cytarabine-Induced Cytotoxicity and Myelosuppression.

Resistance to cytarabine remains a major challenge in the treatment of acute myeloid leukemia (AML). Based on previous studies implicating ABCC4/MRP4 in the transport of nucleosides, we hypothesized that cytarabine is sensitive to ABCC4‐mediated efflux, thereby decreasing its cytotoxic response against AML blasts. The uptake of cytarabine and its monophosphate metabolite was found to be facilitated in ABCC4‐expressing vesicles and intracellular retention was significantly impaired by overexpression of human ABCC4 or mouse Abcc4 (P < 0.05). ABCC4 was expressed highly in AML primary blasts and cell lines, and cytotoxicity of cytarabine in cells was increased in the presence of the ABCC4 inhibitors MK571 or sorafenib, as well as after ABCC4 siRNA. In Abcc4‐null mice, cytarabine‐induced hematological toxicity was enhanced and ex vivo colony‐forming assays showed that Abcc4‐deficiency sensitized myeloid progenitors to cytarabine. Collectively, these studies demonstrate that ABCC4 plays a protective role against cytarabine‐mediated insults in leukemic and host myeloid cells.

Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia

Oncogenic addiction to the Fms-like tyrosine kinase 3 (FLT3) is a hallmark of acute myeloid leukemia (AML) that harbors the FLT3–internal tandem duplication (FLT3-ITD) mutation. While FLT3 inhibitors like sorafenib show initial therapeutic efficacy, resistance rapidly develops through mechanisms that are incompletely understood. Here, we used RNA-Seq–based analysis of patient leukemic cells and found that upregulation of the Tec family kinase BMX occurs during sorafenib resistance. This upregulation was recapitulated in an in vivo murine FLT3-ITD–positive (FLT3-ITD+) model of sorafenib resistance. Mechanistically, the antiangiogenic effects of sorafenib led to increased bone marrow hypoxia, which contributed to HIF-dependent BMX upregulation. In in vitro experiments, hypoxia-dependent BMX upregulation was observed in both AML and non-AML cell lines. Functional studies in human FLT3-ITD+ cell lines showed that BMX is part of a compensatory signaling mechanism that promotes AML cell survival during FLT3 inhibition. Taken together, our results demonstrate that hypoxia-dependent upregulation of BMX contributes to therapeutic resistance through a compensatory prosurvival signaling mechanism. These results also reveal the role of off-target drug effects on tumor microenvironment and development of acquired drug resistance. We propose that the bone marrow niche can be altered by anticancer therapeutics, resulting in drug resistance through cell-nonautonomous microenvironment-dependent effects.

Therapeutic targeting of KDM1A/LSD1 in Ewing sarcoma with SP-2509 engages the endoplasmic reticulum stress response

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 –1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 μmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902–16. ©2018 AACR.

Abstract C170: GDC-0941 inhibits cytarabine-induced PI3K/AKT signaling and promotes synergistic activity in acute myeloid leukemia.

Introduction: We previously identified genes associated with cytarabine (AraC) response in acute myeloid leukemia (AML) and demonstrated increased expression of PIK3C3 to be significantly associated with a detrimental pattern of association (p=0.016) and multiple phenotypes (increased DNA synthesis, increased blast AraC IC50, poor clinical response, and worse event free survival) (Lamba et al., 2011). Here we evaluate the anti-leukemic activity of a selective PI3K inhibitor, GDC-0941, alone and in combination with AraC. Methods and Materials: A panel of 6 AML cell lines were assessed for potential activity of GDC-0941 alone (0.0625-4µM) and in combination with AraC (72h continuous, 4h pulse sequential or simultaneous exposure). We assessed cell viability by MTT assay and implemented the median effect approach to determine if combination drug effects, assessed as the combination index (CI) value, were synergistic, additive, or antagonistic. Induction of apoptosis and alterations in cell cycle were detected by caspase activation and propidium iodide staining, respectively. Western blot analyses were performed to determine drug effects on expression of PI3K isoforms, pAkt, pS6, and pErk signaling. Ex vivo cytotoxicity assays were performed on leukemic blasts isolated from murine primary c-Myc recipients to determine sensitivity to GDC-0941, AraC, and the combination. To evaluate in vivo efficacy, survival studies were performed using a c-Myc induced murine model of AML. Sublethally irradiated (550cGy) syngeneic tertiary recipients were randomized to receive vehicle, GDC-0941 (50mg/kg daily x 5 days, po), AraC (100mg/kg daily x 5 days, ip) or combination (week 1: AraC100mg/kg daily x 5 days; week 2: GDC-0941 50mg/kg daily x 5,) (N=8/treatment arm). Pharmacodynamic assessment of pAkt levels in leukemic blasts among the different treatment arms were also evaluated. Results: GDC-0941 treatment reduced cell viability in a dose-dependent manner and IC50s ranged from 0.36-4µM in AML cell lines. Caspase activity was induced by GDC-0941 alone and enhanced with the combination. For combination treatment, CI values were synergistic in all but 1 cell line (range, 0.20-0.97). Western blot analyses of the U937 cell line revealed that AraC induced both PI3Kα and pAkt signaling which could be abrogated by cotreatment with GDC-0941. Ex vivo combination drug treatments of c-Myc leukemic blasts demonstrated synergistic CI values. Combination treatment significantly prolonged survival in the c-Myc AML model compared to either drug alone or controls (control, median survival=21; GDC-0941, median survival=22; AraC, median survival=26; combination, median survival=28 days; p=0.01 and p=0.03 for AraC vs control and combination vs AraC, respectively) Conclusions: Our results show that AraC mediated induction of PI3Kα and pAkt in AML cells can be augmented by selective targeting of the PI3K/Akt pathway with GDC-0941. From these lines of preclinical evidence, combination of AraC with PI3K inhibitors is a promising strategy to enhance AraC efficacy in AML. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C170. Citation Format: Christina D. Drenberg, Brian Fermanski, Steven Zatechka, Yiping Fan, Shelley Orwick, Laura Janke, Sharyn D. Baker. GDC-0941 inhibits cytarabine-induced PI3K/AKT signaling and promotes synergistic activity in acute myeloid leukemia. [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 C170.