Edwin Chen

Purpose: To characterize an unusual presentation of infectious posterior uveitis using multimodal imaging, and discuss the clinical decision-making involved in diagnosis and treatment. Methods: Wide-field fundus photography, swept-source optical coherence tomography (OCT), swept-source OCT angiography, fluorescein angiography, and indocyanine green angiography. Results: This patient presented with cyclical fevers and blurry vision. Fundus examination revealed bilateral optic disc edema, macular intraretinal white spots and many scattered yellow-white chorioretinal lesions. Multimodal imaging characteristics suggested that many of these lesions represent choroidal granulomas. Extensive systemic workup was only notable for borderline elevated Bartonella henselae IgG titers (1:128), however convalescent IgG titers were elevated at 38 days (1:512) supporting the diagnosis of Bartonella chorioretinitis. Conclusion: Ocular manifestations of Bartonella henselae infection are varied and may include choroidal granulomas. Multimodal imaging characteristics may help identify etiologies of infectious uveitis. Convalescent titers are important when evaluating patients with suspected Bartonellosis, especially patients with atypical presentations.

Calreticulin (CALR) is mutated in the majority of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs). Mutant CALR (CALRdel52) exerts its effect by binding to the thrombopoietin receptor MPL to cause constitutive activation of JAK-STAT signaling. In this study, we performed an extensive mutagenesis screen of the CALR globular N-domain and revealed 2 motifs critical for CALRdel52 oncogenic activity: (1) the glycan-binding lectin motif and (2) the zinc-binding domain. Further analysis demonstrated that the zinc-binding domain was essential for formation of CALRdel52 multimers, which was a co-requisite for MPL binding. CALRdel52 variants incapable of binding zinc were unable to homomultimerize, form CALRdel52-MPL heteromeric complexes, or stimulate JAK-STAT signaling. Finally, treatment with zinc chelation disrupted CALRdel52-MPL complexes in hematopoietic cells in conjunction with preferential eradication of cells expressing CALRdel52 relative to cells expressing other MPN oncogenes. In addition, zinc chelators exhibited a therapeutic effect in preferentially impairing growth of CALRdel52-mutant erythroblasts relative to unmutated erythroblasts in primary cultures of MPN patients. Together, our data implicate zinc as an essential cofactor for CALRdel52 oncogenic activity by enabling CALRdel52 multimerization and interaction with MPL, and suggests that perturbation of intracellular zinc levels may represent a new approach to abrogate the oncogenic activity of CALRdel52 in the treatment of MPNs.

Myeloproliferative neoplasms (MPNs) are a group of haematological malignancies arising from haematopoietic stem cells (HSCs) with acquired driver mutations in JAK2, MPL and CALR. Current therapies are not selective for the mutant HSC population. Increased replication stress is seen in the presence of mutant JAK2, suggesting DNA damage response inhibitors (DDRi) may differentially affect mutant HSCs over wild-type HSCs to restore normal haematopoiesis. Using JAK2V617F and CALR (del 52) mutant cell lines, we observed that ATR inhibition (ATRi) by AZD6738 and VE-821 significantly reduced viability. The combination of ATRi and a hydroxyurea/a JAK1/2 inhibitor—ruxolitinib—demonstrated high synergism in both apoptosis induction and proliferation arrest. This study provides preliminary evidence that ATRi combined with standard therapies may be exploited in MPNs harbouring JAK2 and CALR mutations.

During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.

Key Points: The binding of mutant calreticulin to MPL can be uncoupled from MPL activation. The lectin activity but not the chaperone functionality of mutant CALR is required for cytokine-independent growth.

Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN), but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of patients with CALR-mutant MPN. We further show that the thrombopoietin receptor MPL is required for mutant CALR-driven transformation through JAK–STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN. Significance: The mechanism by which CALR mutations induce MPN remains unknown. In this report, we show that the positive charge of the CALR mutant C-terminus is necessary to transform hematopoietic cells by enabling binding between mutant CALR and the thrombopoietin receptor MPL. Cancer Discov; 6(4); 368–81. ©2016 AACR. See related commentary by Stanley and Steidl, p. 344. This article is highlighted in the In This Issue feature, p. 331

In a mouse model of the 5q- subtype of myelodysplastic syndrome, haploinsufficiency of the ribosomal protein gene Rps14 leads to anemia through a mechanism involving innate immune signaling and the Tlr4 ligand S100A8, which induces a p53-dependent block to erythroid differentiation. Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8—whose expression was increased in mutant erythroblasts, monocytes and macrophages—is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

JAK2 exon 12 mutations are associated with more marked and isolated erythrocytosis than JAK2V617F . We analyzed expression profiles of JAK2 exon 12-mutant and wild-type erythroid colonies from patients with polycythemia vera (PV). Exon 12 mutations were associated with interferon-target gene

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations inJAK2,CALRorMPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance inJAK2^V617F-negative MPN: rs12339666 (JAK2;meta-analysisP=1.27 × 10⁻¹⁰) and rs2201862 (MECOM; meta-analysisP=1.96 × 10⁻⁹). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when includingJAK2^V617F-positive cases. rs9376092 has a stronger effect inJAK2^V617F-negative cases withCALRand/orMPLmutations (Breslow–DayP=4.5 × 10⁻⁷), whereas inJAK2^V617F-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelicχ²P=7.3 × 10⁻⁷). ReducedMYBexpression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences inMYBexpression to disease phenotype.