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Accurate classification of acute myeloid leukaemia (AML) has become increasingly reliant on molecular characterisation of this blood cancer. Throughout Australia and New Zealand massively parallel sequencing (MPS) is being adopted by diagnostic laboratories for the routine evaluation of patients with AML. This technology enables the surveying of many genes simultaneously, with many technical advantages over single gene testing approaches. However, there are many variations in wet and dry lab MPS procedures, which raises the prospect of discordant results between laboratories. This study compared the results obtained from MPS testing of ten diagnostic AML bone marrow aspirate samples sent to eight participating laboratories across Australasia. A reassuringly high concordance of 94% was observed with regard to variant detection and characterisation of pathogenicity. The level of discordance observed, although low, demonstrates the need for ongoing assessment of concordance between diagnostic testing laboratories through quality assurance programs. Refereed/Peer-reviewed

Atypical Behçet’s is recognised in myelodysplastic syndrome (MDS) cases and is associated with trisomy 8. Clonal cytopenia of undetermined significance (CCUS) is recognised as a precursor to MDS. Our case describes the presentation of atypical Behçet’s, in association with CCUS, post a Streptococcal infection. A mutation of a zinc finger RNA spliceosome, ZRSR2, is also described. Our patient initially presented with macrocytic anaemia, together with neutropenia and lymphocytopenia on routine monitoring. Later gastrointestinal symptoms together with oral and anal ulcerations developed. He was treated with oral zinc therapy and had resolution of recurrent oral ulcerations and significant reduction in severity of anal ulcerations. The functional impact of ZRSR2 mutation on spliceosome assembly is yet to be defined, but has been previously reported in CCUS with a clinical phenotype of macrocytic anaemia.

Erythroleukemia (M6 subtype of Acute Myeloid Leukaemia) is uncommon but has a poor prognosis, with reports of successful differentiation therapy using erythropoietin (Epo). Signaling through the Epo-receptor, which involves JAK2 and Lyn tyrosine kinases, controls red blood cell progenitor development. We have highlighted the importance of Lyn for regulating downstream Akt, and feed-back inhibitory signaling of the Epo-receptor through analysis of Lyn-/-, Lynup/up (hyperactive Lyn) and Cbp-/- (Csk-binding protein, a negative regulator of Lyn) erythroid cells. However, the importance of maintaining Lyn activity as opposed to Lyn protein for erythroid cell development and signaling, has not been delineated. To address this, we generated LynKD/KD mice (expressing a kinase dead K275M mutant Lyn), and analysed their erythroid compartment and signaling in immortalized erythroid progenitors. We show that LynKD/KD mice display splenic extramedullary erythropoiesis and have evidence of elevate bone marrow erythropoiesis, similar to Lyn-/- mice but with a less severe phenotype. Immortalized erythroid progenitors from LynKD/KD mice show impaired Epo-induced differentiation and a greater dependence on Epo for viability, but unaltered proliferation, compared to wild-type cells. Epo-induced signaling of LynKD/KD cells showed enhanced pJAK2/pSTAT5, reduced pAkt/pGAB2, and substantially reduced ALAS-e levels, compared to wild-type cells. Importantly, elevating Akt signaling in LynKD/KD cells by addition of phosphatase inhibitors (okadaic acid or Calyculin A), or via expression of active Akt, restored their differentiation capacity (and ALAS-e levels) and reduced their dependence on Epo for viability. We have unveiled that Lyn kinase activity, and not just its expression, is required for correct signaling of Akt to GATA-1 to maintain ALAS-e expression in erythroid cells, enabling hemoglobin production and viability during differentiation. Disclosures No relevant conflicts of interest to declare.

The PR-domain (PRDM) family of genes encodes transcriptional regulators, several of which are deregulated in cancer. By using a functional screening approach, we sought to identify novel tumor suppressors among the PRDMs. Here we demonstrate oncogenic collaboration between depletion of the previously uncharacterized PR-domain family member Prdm11 and overexpression of MYC. Overexpression of PRDM11 inhibits proliferation and induces apoptosis. Prdm11 knockout mice are viable, and loss of Prdm11 accelerates MYC-driven lymphomagenesis in the Eµ-Myc mouse model. Moreover, we show that patients with PRDM11-deficient diffuse large B-cell lymphomas (DLBCLs) have poorer overall survival and belong to the nongerminal center B-cell-like subtype. Mechanistically, genome-wide mapping of PRDM11 binding sites coupled with transcriptome sequencing in human DLBCL cells evidenced that PRDM11 associates with transcriptional start sites of target genes and regulates important oncogenes such as FOS and JUN. Hence, we characterize PRDM11 as a putative novel tumor suppressor that controls the expression of key oncogenes, and we add new mechanistic insight into B-cell lymphomagenesis.

Erythroid homoeostasis is primarily controlled by Epo (erythropoietin) receptor signalling; however, the Lyn tyrosine kinase plays an important subsidiary role in regulating the erythroid compartment. Nonetheless, specific erythroid pathways that require Lyn activity and their biological significance remain unclear. To address this, we asked what consequence loss of Lyn had on the ex vivo expansion and maturation of splenic erythroid progenitors and Epo receptor signalling. Pharmacological inhibition of Lyn with PP2 inhibited the survival of terminally differentiated erythroblasts. Less committed erythroid progenitors expanded well, whereas early splenic Lyn−/− erythroblasts had attenuated ex vivo expansion, and late stage Lyn−/− erythroblasts were retarded in completing morphological maturation ex vivo. Furthermore, immortalized Lyn−/− erythroblasts were slower growing, less viable and inhibited in their differentiation. Signalling studies showed that Lyn was required for both positive GAB2/Akt/FoxO3 (forkhead box O3) survival signals as well as negative feedback of JAK2 (Janus kinase 2)/STAT5 (signal transducer and activator of transcription 5) and ERK1/2 (extracellular-signal-regulated kinase 1/2) signals via SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase 1). During differentiation, Lyn controls survival and cell cycle exit as demonstrated by reduced STAT5 and FoxO3/GSKα/β (glycogen synthase kinase α/β) phosphorylation and diminished p27Kip1 induction in Lyn-deficient erythroblasts. Lyn deficiency alters the balance of pro- and anti-apoptotic molecules (BAD and BclXL), thereby reducing survival and preventing cell cycle exit. Consequently, Lyn facilitates normal erythrocyte production by influencing different stages of erythroid progenitor expansion, and mature cell development and survival signalling.

Erythropoiesis is controlled principally through erythropoietin (Epo) receptor signaling, which involves Janus kinase 2 (JAK2) and Lyn tyrosine kinase, both of which are important for regulating red blood cell (RBC) development. Negative regulation of Lyn involves C-Src kinase (Csk)-mediated phosphorylation of its C-terminal tyrosine, which is facilitated by the transmembrane adaptor Csk-binding protein (Cbp). Although Cbp has significant functions in controlling Lyn levels and activity in erythroid cells in vitro, its importance to primary erythroid cell development and signaling has remained unclear. To address this, we assessed the consequence of loss of Cbp on the erythroid compartment in vivo and whether Epo-responsive cells isolated from Cbp-knockout mice exhibited altered signaling. Our data show that male Cbp−/− mice display a modest but significant alteration to late erythroid development in bone marrow with evidence of increased erythrocytes in the spleen, whereas female Cbp−/− mice exhibit a moderate elevation in early erythroid progenitors (not seen in male mice) that does not influence the later steps in RBC development. In isolated primary erythroid cells and cell lines generated from Cbp−/− mice, survival signaling through Lyn/Akt/FoxO3 was elevated, resulting in sustained viability during differentiation. The high Akt activity disrupted GAB2/SHP-2 feedback inhibition of Lyn; however, the elevated Lyn activity also increased inhibitory signaling via SHP-1 to restrict the Erk1/2 pathway. Interestingly, whereas loss of Cbp led to mild changes to late RBC development in male mice, this was not apparent in female Cbp−/− mice, possibly due to their elevated estrogen, which is known to facilitate early progenitor self-renewal.

Invasion and metastasis are controlled by the invadopodia, which delivers matrix-degrading enzymes to the invasion interface permitting cancer cell penetration and spread into healthy tissue. We have identified a novel pathway that directs Lyn/Src family tyrosine kinase signals to the invadopodia to regulate sarcoma cell invasion via the molecule AFAP-1-like-1 (AFAP1L1), a new member of the AFAP (actin filament-associated protein) family. We show that AFAP1L1 can transform cells, promote migration and co-expression with active Lyn profoundly influences cell morphology and movement. AFAP1L1 intersects several invadopodia pathway components through its multiple domains and motifs, including the following (i) pleckstrin homology domains that bind phospholipids generated at the plasma membrane by phosphoinositide 3-kinase, (ii) a direct filamentous-actin binding domain and (iii) phospho-tyrosine motifs (pY136 and pY566) that specifically bind Vav2 and Nck2 SH2 domains, respectively. These phosphotyrosine motifs are essential for AFAP1L1-mediated cytoskeleton regulation. Through its interaction with Vav2, AFAP1L1 regulates Rac activity and downstream control of PAK1/2/3 (p21-activated kinases) phosphorylation of myosin light chain (MLC) kinase and MLC2. AFAP1L1 interaction with Nck2 recruits actin-nucleating complexes. Significantly, in osteosarcoma cell lines, knockdown of AFAP1L1 inhibits phosphorylated MLC2 recruitment to filamentous-actin structures, disrupts invadopodia formation, cell attachment, migration and invasion. These data define a novel pathway that directs Lyn/Src family tyrosine kinase signals to sarcoma cell invadopodia through specific recruitment of Vav2 and Nck2 to phosphorylated AFAP1L1, to control cell migration and invasion.

Expression of the prostatic tumour suppressor, NKX3.1 is reduced or undetectable in a large proportion of advanced human prostate tumours. Although loss of heterozygosity (LOH) is common at the 8p21.2 locus which encompasses the NKX3.1 gene, mutations of the remaining NKX3.1 allele that would account for loss of NKX3.1 expression have not been identified. Indeed, the documented discordance between NKX3.1 mRNA and protein expression suggests that translational or posttranslational mechanisms contribute to the loss of NKX3.1 expression in prostate tumour tissues. We have identified two novel NKX3.1 binding proteins in LNCaP cells, RMND5A and RMND5B. The RMND5A gene locus at 2p11.1 is deleted or amplified in a variety of cancers, while the RMND5B locus at 5q35.3 undergoes frequent LOH in breast tumours from BRCA1 and BRCA2 mutation carriers and is located within an uncharacterised prostate cancer heritability locus. RMND5 proteins are widely expressed, multidomain proteins that each contain a putative RING domain, suggesting that they are able to function as E3 ubiquitin ligases. In in vitro ubiquitin assays, the RING domain of RMND5A was able to associate with UbcH2, UbcH5b and UbcH5c, while the RING domain of RMND5B interacted with UbcH5b and UbcH5c to mediate ubiquitin transfer. Both RMND5A and RMND5B were associated with ubiquitinated proteins in vivo and the increased expression of RMND5A or RMND5B augmented cellular levels of ubiquitinated proteins, providing further evidence of their E3 ubiquitin ligase activity. Overexpression of either RMND5A or RMND5B in LNCaP cells resulted in dose-dependent declines in endogenous NKX3.1 levels, which were attenuated following proteasome inhibition. In addition, RMND5A or RMND5B overexpression enhanced cellular levels of ubiquitinated NKX3.1. In LNCaP cells, RMND5 proteins were diffusely distributed in the cytoplasm and nucleus, with RMND5B occasionally exhibiting a punctate cytoplasmic distribution. When NKX3.1 and RMND5A or RMND5B were overexpressed, RMND5 proteins became predominantly nuclear, while the nuclear levels of NKX3.1 were markedly reduced. Together, these findings indicate that RMND5 proteins promote NKX3.1 ubiquitination and proteasome-dependent degradation and additionally modify the intracellular localisation of NKX3.1, potentially regulating its function as a transcription factor. Citation Format: Alison Louw, Marc A. Thomas, Jennet Harvey, Jacqueline M. Bentel. Regulation of NKX3.1 by RMND5 proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2013-4075