Elena Bibikova

TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors

Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor-α (TNF-α) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-α or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-α inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-α, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia.

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

Abstract 2596: ACP-196: a novel covalent Bruton's tyrosine kinase (Btk) inhibitor with improved selectivity and in vivo target coverage in chronic lymphocytic leukemia (CLL) patients

Ibrutinib, a first generation Btk inhibitor, is approved for the treatment of CLL and mantle cell lymphoma; known toxicities include atrial fibrillation, diarrhea, rash, arthralgia and bleeding events (1). Recent reports show ibrutinib9s off target effects may negatively impact its potential for combined therapy with anti-CD20 antibodies (2,3). Here we describe the pharmacologic characterization of ACP-196 a potent, novel second generation Btk inhibitor, which binds covalently to Cys481 with improved selectivity and in vivo target coverage. Compared to ibrutinib and CC-292, ACP-196 demonstrated higher selectivity for Btk when profiled against a panel of 395 non-mutant kinases (1 μM) in a competitive binding assay. IC50 determinations on 9 kinases with a Cys in the same position as Btk showed ACP-196 to be the most selective. The improved selectivity is related to the reduced intrinsic reactivity of ACP-1969s electrophile. Importantly, unlike ibrutinib, ACP-196 did not inhibit EGFR, Itk or Txk. Phosphoflow assays on EGFR expressing cell lines confirmed ibrutinib9s EGFR inhibition (EC50: 47-66 nM) with no inhibition observed for ACP-196 at 10 μM. These data may explain the ibrutinib-related incidence of diarrhea and rash. Ibrutinib9s potency on Itk and Txk may explain why it interferes with cell-mediated anti-tumor activities of therapeutic CD20 antibodies and immune-mediated killing in the tumor microenvironment (2,3). In human whole blood, ACP-196 and ibrutinib showed robust and equipotent inhibitory activity on B-cell receptor induced responses in the low nM range, whereas CC-292 was 10-20 fold less potent. In vivo, oral administration of ACP-196 in mice resulted in dose-dependent inhibition of anti-IgM-induced CD86 expression in CD19+ splenocytes with an ED50 of 0.34 mg/kg compared to 0.91 mg/kg for ibrutinib. A similar model was used to compare the duration of Btk inhibition after a single oral dose of 25 mg/kg. ACP-196 and ibrutinib inhibited CD86 expression >90% at 3h and ∼50% at 24h postdose. In contrast, CC-292 inhibited ∼50% at 3h and ∼20% at 24h postdose. An ELISA based Btk target occupancy assay was developed to measure target coverage in preclinical and clinical studies. In healthy volunteers, ACP-196 at an oral dose of 100 mg QD showed >90% target coverage over a 24h period. Btk occupancy and regulation of the PD markers (CD69 and CD86) correlated with PK parameters for exposure. In CLL patients, after 7 days of dosing with ACP-196 at 200 mg QD, 94% Btk target occupancy was observed compared with ∼80% reported for ibrutinib at 420 mg QD (4). In conclusion, ACP-196 is a novel Btk inhibitor with key pharmacologic differentiators versus ibrutinib and CC-292. ACP-196 is currently being evaluated in clinical trials. 1. IMBRUVICA package insert 2014 2. Rajasekaran Blood 2014 Abstr # 3118 3. Da Roit Haematologica 2014 4. Byrd NEJM 2013 Citation Format: Todd Covey, Tjeerd Barf, Michael Gulrajani, Fanny Krantz, Bart van Lith, Elena Bibikova, Bas van de Kar, Edwin de Zwart, Ahmed Hamdy, Raquel Izumi, Allard Kaptein. ACP-196: a novel covalent Bruton9s tyrosine kinase (Btk) inhibitor with improved selectivity and in vivo target coverage in chronic lymphocytic leukemia (CLL) patients. [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 2596. doi:10.1158/1538-7445.AM2015-2596

Abstract 408: ACP-196, an orally bioavailable covalent selective inhibitor of Btk, modulates the innate tumor microenvironment, exhibits antitumor efficacy and enhances gemcitabine activity in pancreatic cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Pancreatic ductal adenocarcinoma exists in a complex desmoplastic microenvironment that provides stromal support for tumor growth and conceals the tumor from immune surveillance. Tumor-associated stroma comprises a mix of fibroblasts, immunosuppressive T regulatory cells (Tregs), myeloid suppressive monocytes (MDSCs) and tumor-associated macrophages (TAMs) that promote tumor growth and restrain immune-mediated tumor cell killing. The targeting of immune infiltrates may impair stromal support and enhance immune-mediated killing of pancreatic cancer cells. Brutons tyrosine kinase (Btk) is a nonreceptor enzyme in the Tec kinase family expressed among cells of hematopoietic origin including B cells, myeloid cells, mast cells and platelets, but not T cells, where it regulates multiple cellular processes. Here we describe an unexpected finding of ACP-196, a potent, novel, second generation Btk inhibitor with improved selectivity and target coverage that binds covalently to a cysteine residue (Cys481) in the front position of the ATP-binding pocket. In an orthotopic mouse model of pancreatic cancer, KPC derived pancreatic cancer cells (KrasG12D; Trp53R172H; Pdx1-Cre) were injected into the pancreases. Vehicle, single agent ACP-196 (15 mg/kg/BID, gavage), single agent gemcitabine (50 mg/kg, IV) and combination ACP-196 with gemcitabine were evaluated for efficacy. By 4 weeks of treatment, mice in the vehicle group showed signs of health deterioration and all mice were euthanized, tumors were collected and measured. Relative to the vehicle treatment, ACP-196 monotherapy resulted in a >2-fold reduction in tumor growth compared with less than a 2-fold reduction with gemcitabine alone. The combination of ACP-196 and gemcitabine resulted in a further reduction in tumor growth when compared to each single agent. Interestingly, analysis of tumor tissues showed that single agent ACP-196 inhibited immunosuppressive populations of TAMs and MDSCs. Surprisingly, Treg populations were also reduced with a robust expansion of CD8+ T cells in the tumors. None of these effects were observed with gemcitabine alone. Although Btk is not expressed in T cells, this finding maybe the result of inhibiting the MDSC and TAM populations within the tumor microenvironment, a mechanism of action which is currently under investigation. Taken together, these data identify Btk as a novel target for modulating tumor immune escape and suggest that pharmacologic targeting of suppressive myeloid cells by ACP-196 induces therapeutic benefit. ACP-196 is currently being evaluated in clinical trials including frontline and salvage pancreatic cancer. Citation Format: Brian J. Lannutti, Michael Gulrajani, Fanny Krantz, Elena Bibikova, Todd Covey, Katti Jessen, Wayne Rothbaum, David M. Johnson, Roger Ulrich. ACP-196, an orally bioavailable covalent selective inhibitor of Btk, modulates the innate tumor microenvironment, exhibits antitumor efficacy and enhances gemcitabine activity in pancreatic cancer. [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 408. doi:10.1158/1538-7445.AM2015-408

Innate immune system activation in zebrafish and cellular models of Diamond Blackfan Anemia

Deficiency of ribosomal proteins (RPs) leads to Diamond Blackfan Anemia (DBA) associated with anemia, congenital defects, and cancer. While p53 activation is responsible for many features of DBA, the role of immune system is less defined. The Innate immune system can be activated by endogenous nucleic acids from non-processed pre-rRNAs, DNA damage, and apoptosis that occurs in DBA. Recognition by toll like receptors (TLRs) and Mda5-like sensors induces interferons (IFNs) and inflammation. Dying cells can also activate complement system. Therefore we analyzed the status of these pathways in RP-deficient zebrafish and found upregulation of interferon, inflammatory cytokines and mediators, and complement. We also found upregulation of receptors signaling to IFNs including Mda5, Tlr3, and Tlr9. TGFb family member activin was also upregulated in RP-deficient zebrafish and in RPS19-deficient human cells, which include a lymphoid cell line from a DBA patient, and fetal liver cells and K562 cells transduced with RPS19 shRNA. Treatment of RP-deficient zebrafish with a TLR3 inhibitor decreased IFNs activation, acute phase response, and apoptosis and improved their hematopoiesis and morphology. Inhibitors of complement and activin also had beneficial effects. Our studies suggest that innate immune system contributes to the phenotype of RPS19-deficient zebrafish and human cells.

Loss of FOXM1 promotes erythropoiesis through increased proliferation of erythroid progenitors.

Forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors and regulates a network of proliferation-associated genes. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor in oncogenesis. FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation, suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord blood CD34+ cells were transduced with FOXM1 short hairpin ribonucleic acid (shRNA) lentivirus. Knockdown of FOXM1 resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally, knockdown of FOXM1 increased bromodeoxyuridine (BrdU) incorporation in erythroid cells, suggesting greater proliferation of erythroid progenitors. We also observed that the defective phosphorylation of FOXM1 by checkpoint kinase 2 (CHK2) or cyclin-dependent kinases 1/2 (CDK1/2) increased the erythroid population in a manner similar to knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, forkhead domain inhibitor-6 (FDI-6), increased red blood cell numbers through increased proliferation of erythroid precursors. Overall, our data suggest a novel function of FOXM1 in normal human hematopoiesis.