PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in human and murine acute myeloid leukemia.

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

DNA Origami Nanostructures Elicit Dose-Dependent Immunogenicity and Are Nontoxic up to High Doses In Vivo.

DNA origami (DO) nanotechnology enables the construction of precise nanostructures capable of functionalization with small molecule drugs, nucleic acids, and proteins, suggesting a promising platform for biomedical applications. Despite the potential for drug and vaccine delivery, the impact of DO vehicles on immunogenicity in vivo is not well understood. Here, two DO vehicles, a flat triangle and a nanorod, at varying concentrations are evaluated in vitro and with a repeated dosing regimen administered at a high dose in vivo to study early and late immunogenicity. The studies show normal CD11b+ myeloid cell populations preferentially internalize DO in vitro. DO structures distribute well systemically in vivo, elicit a modest pro-inflammatory immune response that diminishes over time and are nontoxic as shown by weight, histopathology, lack of cytokine storm, and a complete biochemistry panel at the day 10 end point. The results take critical steps to characterize the biological response to DO and suggest that DO vehicles represent a promising platform for drug delivery and vaccine development where immunogenicity should be a key consideration.

Leukemic B Cell CTLA-4 Suppresses Costimulation of T Cells.

The clinical benefit of CTLA-4 blockade on T cells is known, yet the impact of its expression on cancer cells remains unaddressed. We define an immunosuppressive role for tumor-expressed CTLA-4 using chronic lymphocytic leukemia (CLL) as a disease model. CLL cells, among other cancer cells, are CTLA-4+ Coculture with activated human T cells induced surface CTLA-4 on primary human CLL B cells. CTLA-4 on CLL-derived human cell lines decreased CD80 expression on cocultured CD80+ cells, with restoration upon CTLA-4 blockade. Coculture of CTLA-4+ CLL cells with CD80-GFP+ cell lines revealed transfer of CD80-GFP into CLL tumor cells, similar to CTLA-4+ T cells able to trans-endocytose CD80. Coculture of T cells with CTLA-4+ CLL cells decreased IL-2 production. Using a human CTLA-4 knock-in mouse lacking FcγR function, antitumor efficacy was observed by blocking murine CTLA-4 on tumor cells in isolation of the T cell effect and Fc-mediated depletion. These data implicate tumor CTLA-4 in cancer cell-mediated immunosuppression in vitro and as having a functional role in tumor cells in vivo.

TCL1 targeting miR-3676 is codeleted with tumor protein p53 in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia and dysregulation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease based on transgenic mouse studies. To determine a role of microRNAs on the pathogenesis of the aggressive form of CLL we studied regulation of TCL1 expression in CLL by microRNAs. We identified miR-3676 as a regulator of TCL1 expression. We demonstrated that miR-3676 targets three consecutive 28-bp repeats within 3'UTR of TCL1 and showed that miR-3676 is a powerful inhibitor of TCL1. We further showed that miR-3676 expression is significantly down-regulated in four groups of CLL carrying the 11q deletions, 13q deletions, 17p deletions, or a normal karyotype compared with normal CD19(+) cord blood and peripheral blood B cells. In addition, the sequencing of 539 CLL samples revealed five germ-line mutations in six samples (1%) in miR-3676. Two of these mutations were loss-of-function mutations. Because miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 (Tp53), and is codeleted with Tp53, we propose that loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression.

OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells.

Aberrant regulation of endogenous survival pathways plays a major role in progression of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors within the tumor environmental niche activates survival and proliferation pathways in CLL. Of these, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway appears to be pivotal to support CLL pathogenesis, and pharmacologic inhibitors targeting this axis have shown clinical activity. Here we investigate OSU-T315, a compound that disrupts the PI3K/AKT pathway in a novel manner. Dose-dependent selective cytotoxicity by OSU-T315 is noted in both CLL-derived cell lines and primary CLL cells relative to normal lymphocytes. In contrast to the highly successful Bruton's tyrosine kinase and PI3K inhibitors that inhibit B-cell receptor (BCR) signaling pathway at proximal kinases, OSU-T315 directly abrogates AKT activation by preventing translocation of AKT into lipid rafts without altering the activation of receptor-associated kinases. Through this mechanism, the agent triggers caspase-dependent apoptosis in CLL by suppressing BCR, CD49d, CD40, and Toll-like receptor 9-mediated AKT activation in an integrin-linked kinase-independent manner. In vivo, OSU-T315 attains pharmacologically active drug levels and significantly prolongs survival in the TCL1 mouse model. Together, our findings indicate a novel mechanism of action of OSU-T315 with potential therapeutic application in CLL.

Fcγ receptor-induced soluble vascular endothelial growth factor receptor-1 (VEGFR-1) production inhibits angiogenesis and enhances efficacy of anti-tumor antibodies.

Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

Inhibition of Enhancer of Zeste Homolog 2 Induces Blast Differentiation, Impairs Engraftment and Prolongs Survival in Murine Models of Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. METHODS: The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. RESULTS: EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. CONCLUSIONS: Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

Pyrimidine depletion enhances targeted and immune therapy combinations in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765.

B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in knockout mouse models its mutation has a relatively B cell-specific phenotype. Herein, we demonstrate that BTK protein and mRNA are significantly over expressed in CLL compared with normal B cells. Although BTK is not always constitutively active in CLL cells, BCR or CD40 signaling is accompanied by effective activation of this pathway. Using the irreversible BTK inhibitor PCI-32765, we demonstrate modest apoptosis in CLL cells that is greater than that observed in normal B cells. No influence of PCI-32765 on T-cell survival is observed. Treatment of CD40 or BCR activated CLL cells with PCI-32765 results in inhibition of BTK tyrosine phosphorylation and also effectively abrogates downstream survival pathways activated by this kinase including ERK1/2, PI3K, and NF-κB. In addition, PCI-32765 inhibits activation-induced proliferation of CLL cells in vitro, and effectively blocks survival signals provided externally to CLL cells from the microenvironment including soluble factors (CD40L, BAFF, IL-6, IL-4, and TNF-α), fibronectin engagement, and stromal cell contact. Based on these collective data, future efforts targeting BTK with the irreversible inhibitor PCI-32765 in clinical trials of CLL patients is warranted.

Combination anti-CD74 (milatuzumab) and anti-CD20 (rituximab) monoclonal antibody therapy has in vitro and in vivo activity in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a median survival of 3 years despite chemoimmunotherapy. Rituximab, a chimeric anti-CD20 monoclonal antibody (mAb), has shown only modest activity as single agent in MCL. The humanized mAb milatuzumab targets CD74, an integral membrane protein linked with promotion of B-cell growth and survival, and has shown preclinical activity against B-cell malignancies. Because rituximab and milatuzumab target distinct antigens and potentially signal through different pathways, we explored a preclinical combination strategy in MCL. Treatment of MCL cell lines and primary tumor cells with immobilized milatuzumab and rituximab resulted in rapid cell death, radical oxygen species generation, and loss of mitochondrial membrane potential. Cytoskeletal distrupting agents significantly reduced formation of CD20/CD74 aggregates, cell adhesion, and cell death, highlighting the importance of actin microfilaments in rituximab/milatuzumab-mediated cell death. Cell death was independent of caspase activation, Bcl-2 family proteins or modulation of autophagy. Maximal inhibition of p65 nuclear translocation was observed with combination treatment, indicating disruption of the NF-κB pathway. Significant in vivo therapeutic activity of combination rituximab and milatuzumab was demonstrated in a preclinical model of MCL. These data support clinical evaluation of combination milatuzumab and rituximab therapy in MCL.

Mass Cytometry as a Tool for Investigating Senescence in Multiple Model Systems.

Cellular senescence is a durable cell cycle arrest as a result of the finite proliferative capacity of cells. Senescence responds to both intrinsic and extrinsic cellular stresses, such as aging, mitochondrial dysfunction, irradiation, and chemotherapy. Here, we report on the use of mass cytometry (MC) to analyze multiple model systems and demonstrate MC as a platform for senescence analysis at the single-cell level. We demonstrate changes to p16 expression, cell cycling fraction, and histone tail modifications in several established senescent model systems and using isolated human T cells. In bone marrow mesenchymal stromal cells (BMSCs), we show increased p16 expression with subsequent passage as well as a reduction in cycling cells and open chromatin marks. In WI-38 cells, we demonstrate increased p16 expression with both culture-induced senescence and oxidative stress-induced senescence (OSIS). We also use Wanderlust, a trajectory analysis tool, to demonstrate how p16 expression changes with histone tail modifications and cell cycle proteins. Finally, we demonstrate that repetitive stimulation of human T cells with CD3/CD28 beads induces an exhausted phenotype with increased p16 expression. This p16-expressing population exhibited higher expression of exhaustion markers such as EOMES and TOX. This work demonstrates that MC is a useful platform for studying senescence at a single-cell protein level, and is capable of measuring multiple markers of senescence at once with high confidence, thereby improving our understanding of senescent pathways.

Milatuzumab immunoliposomes induce cell death in CLL by promoting accumulation of CD74 on the surface of B cells.

Chronic lymphocytic leukemia (CLL) is an incurable progressive disease for which new therapies are required. Therapy with monoclonal antibodies (mAbs) has improved the outcome of patients with CLL, making further investigation of novel antibodies directed against alternative and specific targets on B cells an important area of translational research. We now describe functional properties of an antagonistic humanized mAb to CD74, milatuzumab, showing that milatuzumab combined with a crosslinking antibody induces cytotoxicity in vitro in CLL cells in a caspase- and stromal-independent manner associated with aggregation of CD74 on the cell surface. Furthermore, incorporation of milatuzumab into an immunoliposome induces even more of a cytotoxic response than in vitro crosslinking, representing a novel therapeutic formulation for this mAb. Based on these data, future development of the milatuzumab-immunoliposome formulation as a therapeutic agent for CLL is warranted.

17-DMAG targets the nuclear factor-kappaB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition.

The HSP90 client chaperone interaction stabilizes several important enzymes and antiapoptotic proteins, and pharmacologic inhibition of HSP90 results in rapid client protein degradation. Therefore, HSP90 inhibition is an attractive therapeutic approach when this protein is active, a phenotype commonly observed in transformed but not normal cells. However, preclinical studies with HSP90 inhibitors such as 17-AAG demonstrated depletion of only a subset of client proteins and very modest tumor cytotoxicity in chronic lymphocytic leukemia (CLL) cells. Herein, we describe another HSP90 inhibitor, 17-DMAG, which is cytotoxic to CLL but not normal lymphocytes. Treatment with 17-DMAG leads to depletion of the HSP90 client protein IKK, resulting in diminished NF-kappaB p50/p65 DNA binding, decreased NF-kappaB target gene transcription, and caspase-dependent apoptosis. Furthermore, treatment with 17-DMAG significantly decreased the white blood cell count and prolonged the survival in a TCL1-SCID transplant mouse model. The ability of 17-DMAG to function as an NF-kappaB inhibitor is of great interest clinically, as few currently available CLL drugs target this transcription factor. Therefore, the effect of 17-DMAG on NF-kappaB signaling pathways represents a novel therapy warranting further clinical pursuit in this and other B-cell lymphoproliferative disorders.

Epigenetic changes during disease progression in a murine model of human chronic lymphocytic leukemia.

Epigenetic alterations, including gain or loss of DNA methylation, are a hallmark of nearly every malignancy. Changes in DNA methylation can impact expression of cancer-related genes including apoptosis regulators and tumor suppressors. Because such epigenetic changes are reversible, they are being aggressively investigated as potential therapeutic targets. Here we use the Emu-TCL1 transgenic mouse model of chronic lymphocytic leukemia (CLL) to determine the timing and patterns of aberrant DNA methylation, and to investigate the mechanisms that lead to aberrant DNA methylation. We show that CLL cells from Emu-TCL1 mice at various stages recapitulate epigenetic alterations seen in human CLL. Aberrant methylation of promoter sequences is observed as early as 3 months of age in these animals, well before disease onset. Abnormally methylated promoter regions include binding sites for the transcription factor FOXD3. We show that loss of Foxd3 expression due to an NF-kappaB p50/p50:HDAC1 repressor complex occurs in TCL1-positive B cells before methylation. Therefore, specific transcriptional repression is an early event leading to epigenetic silencing of target genes in murine and human CLL. These results provide strong rationale for the development of strategies to target NF-kappaB components in CLL and potentially other B-cell malignancies.

TP-0903 Is Active in Preclinical Models of Acute Myeloid Leukemia with TP53 Mutation/Deletion.

Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene TP53 confers a dismal prognosis with 3-year overall survival of <5%. While inhibition of kinases involved in cell cycle regulation induces synthetic lethality in a variety of TP53 mutant cancers, this strategy has not been evaluated in mutant TP53 AML. Previously, we demonstrated that TP-0903 is a novel multikinase inhibitor with low nM activity against AURKA/B, Chk1/2, and other cell cycle regulators. Here, we evaluated the preclinical activity of TP-0903 in TP53 mutant AML cell lines, including a single-cell clone of MV4-11 containing a TP53 mutation (R248W), Kasumi-1 (R248Q), and HL-60 (TP 53 null). TP-0903 inhibited cell viability (IC50, 12−32 nM) and induced apoptosis at 50 nM. By immunoblot, 50 nM TP-0903 upregulated pChk1/2 and pH2AX, suggesting induction of DNA damage. The combination of TP-0903 and decitabine was additive in vitro, and in vivo significantly prolonged median survival compared to single-agent treatments in mice xenografted with HL-60 (vehicle, 46 days; decitabine, 55 days; TP-0903, 63 days; combination, 75 days) or MV4-11 (R248W) (51 days; 62 days; 81 days; 89 days) (p < 0.001). Together, these results provide scientific premise for the clinical evaluation of TP-0903 in combination with decitabine in TP53 mutant AML.

Molecular, clinical, and prognostic implications of PTPN11 mutations in acute myeloid leukemia.

Prognostic factors associated with chemotherapy outcomes in patients with acute myeloid leukemia (AML) are extensively reported, and one gene whose mutation is recognized as conferring resistance to several newer targeted therapies is protein tyrosine phosphatase non-receptor type 11 (PTPN11). The broader clinical implications of PTPN11 mutations in AML are still not well understood. The objective of this study was to determine which cytogenetic abnormalities and gene mutations co-occur with PTPN11 mutations and how PTPN11 mutations affect outcomes of patients treated with intensive chemotherapy. We studied 1725 patients newly diagnosed with AML (excluding acute promyelocytic leukemia) enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. In 140 PTPN11-mutated patient samples, PTPN11 most commonly co-occurred with mutations in NPM1, DNMT3A, and TET2. PTPN11 mutations were relatively common in patients with an inv(3)(q21q26)/t(3;3)(q21;q26) and a normal karyotype but were very rare in patients with typical complex karyotype and core-binding factor AML. Mutations in the N-terminal SH2 domain of PTPN11 were associated with a higher early death rate than those in the phosphatase domain. PTPN11 mutations did not affect outcomes of NPM1-mutated patients, but these patients were less likely to have co-occurring kinase mutations (ie, FLT3-ITD), suggesting activation of overlapping signaling pathways. However, in AML patients with wild-type NPM1, PTPN11 mutations were associated with adverse patient outcomes, providing a rationale to study the biology and treatment approaches in this molecular group. This trial was registered at www.clinicaltrials.gov as #NCT00048958 (CALGB 8461), #NCT00899223 (CALGB 9665), and #NCT00900224 (CALGB 20202).

Preclinical evaluation of the Hsp90 inhibitor SNX-5422 in ibrutinib resistant CLL.

B-cell receptor (BCR) antagonists such as the BTK inhibitor ibrutinib have proven to effectively target chronic lymphocytic leukemia (CLL) tumor cells, leading to impressive response rates in these patients. However patients do still relapse on ibrutinib, and the progressive disease is often quite aggressive requiring immediate treatment. Several strategies are being pursued to treat patients who relapse on ibrutinib therapy. As the most common form of relapse is the development of a mutant form of BTK which limits ibrutinib binding, agents which lead to degradation of the BTK protein are a promising strategy. Our study explores the efficacy of the Hsp90 inhibitor, SNX-5422, in CLL. The SNX Hsp90 inhibitor was effective in primary CLL cells, as well as B-cell lines expressing either BTK wild type or C481 mutant BTK, which has been identified as the primary resistance mechanism to ibrutinib in CLL patients. Furthermore the combination of SNX-5422 and ibrutinib provided a remarkable in vivo survival benefit in the Eµ-TCL1 mouse model of CLL compared to the vehicle or single agent groups (51 day median survival in the vehicle and ibrutinib groups versus 100 day median survival in the combination). We report here preclinical data suggesting that the Hsp90 inhibitor SNX-5422, which has been pursued in clinical trials in both solid tumor and hematological malignancies, is a potential therapy for ibrutinib resistant CLL.

NF-kappaB regulation of YY1 inhibits skeletal myogenesis through transcriptional silencing of myofibrillar genes.

NF-kappaB signaling is implicated as an important regulator of skeletal muscle homeostasis, but the mechanisms by which this transcription factor contributes to muscle maturation and turnover remain unclear. To gain insight into these mechanisms, gene expression profiling was examined in C2C12 myoblasts devoid of NF-kappaB activity. Interestingly, even in proliferating myoblasts, the absence of NF-kappaB caused the pronounced induction of several myofibrillar genes, suggesting that NF-kappaB functions as a negative regulator of late-stage muscle differentiation. Although several myofibrillar promoters contain predicted NF-kappaB binding sites, functional analysis using the troponin-I2 gene as a model revealed that NF-kappaB-mediated repression does not occur through direct DNA binding. In the search for an indirect mediator, the transcriptional repressor YinYang1 (YY1) was identified. While inducers of NF-kappaB stimulated YY1 expression in multiple cell types, genetic ablation of the RelA/p65 subunit of NF-kappaB in both cultured cells and adult skeletal muscle correlated with reduced YY1 transcripts and protein. NF-kappaB regulation of YY1 occurred at the transcriptional level, mediated by direct binding of the p50/p65 heterodimer complex to the YY1 promoter. Furthermore, YY1 was found associated with multiple myofibrillar promoters in C2C12 myoblasts containing NF-kappaB activity. Based on these results, we propose that NF-kappaB regulation of YY1 and transcriptional silencing of myofibrillar genes represent a new mechanism by which NF-kappaB functions in myoblasts to modulate skeletal muscle differentiation.