DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

Activated granulocytes and inflammatory cytokine signaling drive T-cell lymphoma progression and disease symptoms.

Peripheral T-cell lymphomas (PTCLs), especially angioimmunoblastic and follicular TCLs, have a dismal prognosis because of the lack of efficient therapies, and patients' symptoms are often dominated by an inflammatory phenotype, including fever, night sweats, weight loss, and skin rash. In this study, we investigated the role of inflammatory granulocytes and activated cytokine signaling on T-cell follicular helper-type PTCL (TFH-PTCL) disease progression and symptoms. We showed that ITK-SYK-driven murine PTCLs and primary human TFH-PTCL xenografts both induced inflammation in mice, including murine neutrophil expansion and massive cytokine release. Granulocyte/lymphoma interactions were mediated by positive autoregulatory cytokine loops involving interferon gamma (CD4+ malignant T cells) and interleukin 6 (IL-6; activated granulocytes), ultimately inducing broad JAK activation (JAK1/2/3 and TYK2) in both cell types. Inflammatory granulocyte depletion via antibodies (Ly6G), genetic granulocyte depletion (LyzM-Cre/MCL1flox/flox), or IL-6 deletion within microenvironmental cells blocked inflammatory symptoms, reduced lymphoma infiltration, and enhanced mouse survival. Furthermore, unselective JAK inhibitors (ruxolitinib) inhibited both TCL progression and granulocyte activation in various PTCL mouse models. Our results support the important role of granulocyte-driven inflammation, cytokine-induced granulocyte/CD4+ TCL interactions, and an intact JAK/STAT signaling pathway for TFH-PTCL development and also support broad JAK inhibition as an effective treatment strategy in early disease stages.

Human erythroleukemia genetics and transcriptomes identify master transcription factors as functional disease drivers.

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

Questions and Controversies in the Clinical Application of Tyrosine Kinase Inhibitors to Treat Patients with Radioiodine-Refractory Differentiated Thyroid Carcinoma: Expert Perspectives.

Notwithstanding regulatory approval of lenvatinib and sorafenib to treat radioiodine-refractory differentiated thyroid carcinoma (RAI-R DTC), important questions and controversies persist regarding this use of these tyrosine kinase inhibitors (TKIs). RAI-R DTC experts from German tertiary referral centers convened to identify and explore such issues; this paper summarizes their discussions. One challenge is determining when to start TKI therapy. Decision-making should be shared between patients and multidisciplinary caregivers, and should consider tumor size/burden, growth rate, and site(s), the key drivers of RAI-R DTC morbidity and mortality, along with current and projected tumor-related symptomatology, co-morbidities, and performance status. Another question involves choice of first-line TKIs. Currently, lenvatinib is generally preferred, due to greater increase in progression-free survival versus placebo treatment and higher response rate in its pivotal trial versus that of sorafenib; additionally, in those studies, lenvatinib but not sorafenib showed overall survival benefit in subgroup analysis. Whether recommended maximum or lower TKI starting doses better balance anti-tumor effects versus tolerability is also unresolved. Exploratory analyses of lenvatinib pivotal study data suggest dose-response effects, possibly favoring higher dosing; however, results are awaited of a prospective comparison of lenvatinib starting regimens. Some controversy surrounds determination of net therapeutic benefit, the key criterion for continuing TKI therapy: if tolerability is acceptable, overall disease control may justify further treatment despite limited but manageable progression. Future research should assess potential guideposts for starting TKIs; fine-tune dosing strategies and further characterize antitumor efficacy; and evaluate interventions to prevent and/or treat TKI toxicity, particularly palmar-plantar erythrodysesthesia and fatigue.

Optimized Xenograft Protocol for Chronic Lymphocytic Leukemia Results in High Engraftment Efficiency for All CLL Subgroups.

Preclinical drug development for human chronic lymphocytic leukemia (CLL) requires robust xenograft models recapitulating the entire spectrum of the disease, including all prognostic subgroups. Current CLL xenograft models are hampered by inefficient engraftment of good prognostic CLLs, overgrowth with co-transplanted T cells, and the need for allogeneic humanization or irradiation. Therefore, we aimed to establish an effective and reproducible xenograft protocol which allows engraftment of all CLL subtypes without the need of humanization or irradiation. Unmanipulated NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac (NOG) mice in contrast to C.Cg-Rag2tm1Fwa-/-Il2rgtm1Sug/JicTac (BRG) mice allowed engraftment of all tested CLL subgroups with 100% success rate, if CLL cells were fresh, injected simultaneously intra-peritoneally and intravenously, and co-transferred with low fractions of autologous T cells (2%-4%). CLL transplanted NOG mice (24 different patients) developed CLL pseudofollicles in the spleen, which increased over 4-6 weeks, and were then limited by the expanding autologous T cells. Ibrutinib treatment studies were performed to validate our model, and recapitulated treatment responses seen in patients. In conclusion, we developed an easy-to-use CLL xenograft protocol which allows reliable engraftment for all CLL subgroups without humanization or irradiation of mice. This protocol can be widely used to study CLL biology and to explore novel drug candidates.

Microenvironmental stromal cells abrogate NF-κB inhibitor-induced apoptosis in chronic lymphocytic leukemia.

Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) is known to play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Several NF-κB inhibitors were shown to successfully induce apoptosis of CLL cells in vitro Since the microenvironment is known to be crucial for the survival of CLL cells, herein, we tested whether NF-κB inhibition may still induce apoptosis in these leukemic cells in the presence of protective stromal interaction. We used the specific NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ). Microenvironmental support was mimicked by co-culturing CLL cells with bone marrow-derived stromal cell lines (HS-5 and M2-10B4). NF-κB inhibition by DHMEQ in CLL cells could be confirmed in both the monoculture and co-culture setting. In line with previous reports, NF-κB inhibition induced apoptosis in the monoculture setting by activating the intrinsic apoptotic pathway resulting in poly (ADP-ribose) polymerase (PARP)-cleavage; however, it was unable to induce apoptosis in leukemic cells co-cultured with stromal cells. Similarly, small interfering ribonucleic acid (siRNA)-mediated RELA downregulation induced apoptosis of CLL cells cultured alone, but not in the presence of supportive stromal cells. B-cell activating factor (BAFF) was identified as a microenvironmental messenger potentially protecting the leukemic cells from NF-κB inhibition-induced apoptosis. Finally, we show improved sensitivity of stroma-supported CLL cells to NF-κB inhibition when combining the NF-κB inhibitor with the SYK inhibitor R406 or the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, agents known to inhibit the stroma-leukemia crosstalk. We conclude that NF-κB inhibitors are not promising as monotherapies in CLL, but may represent attractive therapeutic partners for ibrutinib and R406.

Premature terminal differentiation protects from deregulated lymphocyte activation by ITK-Syk.

The development of hematopoietic neoplasms is often associated with mutations, altered gene expression or chromosomal translocations. Recently, the t(5, 9)(q33;q22) translocation was found in a subset of peripheral T cell lymphomas and was shown to result in an IL-2-inducible kinase-spleen tyrosine kinase (ITK-Syk) fusion transcript. In this study, we show that T cell-specific expression of the ITK-Syk oncogene in mice leads to an early onset and aggressive polyclonal T cell lymphoproliferation with concomitant B cell expansion and systemic inflammation by 7-9 wk of age. Because this phenotype is strikingly different from previous work showing that ITK-Syk expression causes clonal T cell lymphoma by 20-27 wk of age, we investigated the underlying molecular mechanism in more detail. We show that the reason for the severe phenotype is the lack of B-lymphocyte-induced maturation protein-1 (Blimp-1) induction by low ITK-Syk expression. In contrast, high ITK-Syk oncogene expression induces terminal T cell differentiation in the thymus by activating Blimp-1, thereby leading to elimination of oncogene-expressing cells early in development. Our data suggest that terminal differentiation is an important mechanism to prevent oncogene-expressing cells from malignant transformation, as high ITK-Syk oncogene activity induces cell elimination. Accordingly, for transformation, a specific amount of oncogene is required, or alternatively, the induction of terminal differentiation is defective.

Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors.

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

Insulin-like growth factor-1 receptor (IGF1R) as a novel target in chronic lymphocytic leukemia.

The receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) is implicated in various tumor entities including chronic lymphocytic leukemia (CLL), but its functional significance in this disease remains poorly characterized. Here, we show that the IGF1R protein is overexpressed in various CLL subsets, suggesting a contribution to CLL pathology. Indeed, we show that IGF1R knockdown in primary human CLL cells compromised their viability. Likewise, IGF1R inhibition with 3 structurally distinct compounds induced apoptosis, even in the presence of protective stroma components. Furthermore, IGF1R inhibition effectively limited CLL development in Eµ-TCL1 transgenic mice and of primary human CLL xenografts. In agreement with its prosurvival function, IGF1R inhibition affected the phosphorylation and/or expression of multiple signaling proteins. The multikinase inhibitor sorafenib yielded similar effects on these signaling elements as IGF1R inhibitors. Indeed, IGF1R appears to be a direct sorafenib target because sorafenib decreased IGF1R expression and phosphorylation, counteracted insulin-like growth factor-1 (IGF-1) binding to CLL cells, and lowered the in vitro kinase activity of recombinant, purified IGF1R. Thus, we demonstrate that blockade of IGF1R-mediated signaling represents a novel mechanism of action for sorafenib in CLL. Importantly, IGF1R inhibitors compromise CLL viability in their microenvironment context, implicating this RTK as a promising therapeutic target.