'Nothing beats the doctor's face to impart trust in their judgement': the role of telehealth in cancer care.

Despite Western Australia having low COVID-19 case numbers and limited community transmission, cancer service delivery changes were introduced early in the pandemic, including adoption of telehealth. Patients attending telehealth appointments during COVID-19 between 11 May 2020 and 7 August 2020 reported that telehealth lessened their concerns and met their needs to varying degrees. Despite this, 56% of patients still preferred in-person appointments.

A cross-sectional analysis of out-of-pocket expenses for people living with a cancer in rural and outer metropolitan Western Australia.

Objective To determine the extent of medical and non-medical out-of-pocket expenses (OOPE) among regional/rural and outer metropolitan Western Australian patients diagnosed with cancer, and the factors associated with higher costs. Methods Cross-sectional data were collected from adult patients living in four regional/rural areas and two outer metropolitan regions in Western Australia who had been diagnosed with breast, prostate, colorectal or lung cancer. Consenting participants were mailed demographic and financial questionnaires, and requested to report all OOPE related to their cancer treatment. Results The median total OOPE reported by 308 regional/rural participants and 119 outer metropolitan participants were A$1518 (interquartile range (IQR): A$581-A$3769) and A$2855 (IQR: A$958-A$7142) respectively. Participants most likely to experience higher total OOPE were younger than 65 years of age, male, resided in the outer metropolitan area, worked prior to diagnosis, had private health insurance, were in a relationship, and underwent surgery. Multivariate analysis of regional/rural participants revealed that receiving care at a rural cancer centre was associated with significantly lower non-medical OOPE (estimated mean A$805, 95% confidence interval (CI): A$735-A$875, P=0.038; compared with other rural participants (A$1347, 95% CI: A$743-A$1951, P<0.001)). Conclusion The cancer patients who participated in this study experienced variation in OOPE, with outer metropolitan participants reporting higher OOPE compared with their regional/rural counterparts. There is a need for cost transparency and access to care close to home, so that patients can make informed choices about where to receive their care. What is known about the topic? In recent years, OOPE for health care in general and cancer in particular have been widely debated by consumers and not-for-profit organisations; the topic has attracted much political attention because it affects both equity and access to care and has wider financial implications for the community. Research studies and reports from both consumer organisations and a Ministerial Advisory Committee found that cancer patients can face exorbitant out-of-pocket costs, and that individuals with private health insurance and those with prostate and breast cancer reported higher costs. In Western Australia, a cancer centre providing comprehensive cancer care was established in the second most populous region to ameliorate the high costs for travel and accommodation that regional cancer patients are known to experience. What does this paper add? This study is unique because it collected detailed cost information from patients and reports on the OOPE of regional/rural and outer metropolitan Western Australian patients receiving care for one of the four most common cancers; it therefore offers novel insight into the experiences of these groups. This study demonstrates that outer metropolitan cancer patients are experiencing much higher OOPE compared with regional/rural cancer patients. Additionally, regional/rural study participants who accessed a Regional Cancer Centre experienced significantly lower non-medical OOPE, compared with regional/rural study participants receiving care elsewhere. What are the implications for practitioners? First, there is a need for improved communication of OOPE to minimise costs to the patient, for example, by facilitating access to local cancer care. Health service providers and insurance companies can improve cost transparency for cancer patients by making this information more readily available, allowing patients to make informed financial choices about where to seek care. Second, the needs of working patients deserve specific attention. These patients face significant work uncertainty and additional distress following a cancer diagnosis.

'Biggest factors in having cancer were costs and no entitlement to compensation'-The determinants of out-of-pocket costs for cancer care through the lenses of rural and outer metropolitan Western Australians.

OBJECTIVE: The aim of this qualitative analysis was to determine patients' perceptions of the impact of cancer-related costs. DESIGN: A qualitative inductive content analysis of the comment sections of surveys completed by participants in a cross-sectional study of cancer-related expenses. SETTING: Residents of 4 regional/rural and 2 outer metropolitan areas in Western Australia. PARTICIPANTS: Adults diagnosed with breast, prostate, colorectal or lung cancer participated in the study between 1 April 2014 and 31 April 2017. MAIN OUTCOME MEASURES: This study identified the key factors contributing to the cost experiences reported by the participants. RESULTS: Participant comments were organised into 4 main categories perceived to incorporate the key factors contributing to the cost experiences reported by 300 participants: 1) health care system factors (access to care in the public or private sector, availability of services close to home, gap payments, cost of travel) 2) financial factors (impact of cancer on employment, ability to work, and career; and strategies for improving financial difficulties) 3) social and community support provided by the government and not-for-profit organisations and 4) understanding of the health care system. CONCLUSION: There is a need for cost transparency, initiatives for no- or low-fee billing providers, access to care close to home, development of financial assistance schemes and return to work programs to alleviate the financial hardship experienced by cancer patients and their families.

'I had to tell my GP I had lung cancer': patient perspectives of hospital- and community-based lung cancer care.

Lung cancer care spans both hospital- and community-based healthcare settings, and suboptimal communication between healthcare providers impacts on continuity and quality of care. Patients' experiences regarding: (1) communication between healthcare providers; and (2) the role of their GP during cancer treatment was explored in interviews with 47 Western Australian lung cancer patients. Thematic analysis using a phenomenological approach was undertaken to derive key themes regarding participant experiences. Poor communication between hospital cancer specialists (HCSs) influenced participants' treatment choices and perceptions of the quality of their care. Information provided by HCSs to GPs was often delayed or incomplete, and many participants perceived themselves as a messenger between healthcare settings. Participants' opinions about the GP role during cancer treatment ranged from 'no role' to an 'active member of the treating team'. A new model of cancer care is needed where the GP is considered part of the treating team. Early involvement of GPs and two-way communication between hospital and primary care during the disease continuum is required for this to occur.

' If I don't have that sort of money again, what happens?': adapting a qualitative model to conceptualise the consequences of out-of-pocket expenses for cancer patients in mixed health systems.

Objective The aim of this study was to explore Western Australian cancer patients' experiences of out-of-pocket expenses (OOPE) during diagnosis and cancer treatment using a phenomenological approach. Methods Semi-structured interviews were conducted with a purposive convenience sample of 40 Western Australian cancer patients diagnosed with breast, lung, prostate or colorectal cancer. Participants were asked about the impact of their diagnosis, the associated costs and their experience within the health system. Data were analysed using thematic content analysis. Results Three key themes influencing participant OOPE experiences were identified: (1) personal circumstances; (2) communication with health providers; and (3) coping strategies. Despite Australia's public healthcare system, several participants found the costs affected their financial security and resorted to coping strategies including medication rationing and restrictive household budgeting. The key themes had a complex and interrelated effect on patient OOPE experiences and were used to adapt Carrera et al.'s model of economic consequences of cancer treatment on the patient and patient coping to describe these relationships in a mixed healthcare system. Conclusion Organised efforts must be implemented to mitigate maladaptive coping strategies being used by cancer patients: (1) health providers should seek informed financial consent from patients before commencing treatment; and (2) financial aid and support schemes for cancer patients should be reviewed to ensure they are delivered equitably. What is known on this topic? The financial cost of cancer can have significant adverse effects on cancer patients. Although financial transparency is desired by cancer patients, its implementation in practice is not clear. What does this paper add? This study adapts a conceptual model for the economic consequences of a cancer diagnosis and repurposes it for a mixed public-private health system, providing a framework for understanding downstream consequences of cancer costs and highlighting opportunities for intervention. What are the implications for health practitioners? Health practitioners need to initiate discussions concerning treatment costs earlier with cancer patients. There are several resources and guides available to assist and facilitate financial transparency. Without urgent attention to the financial consequences of cancer treatment and related expenses, we continue to leave patients at risk of resorting to maladaptive coping strategies, such as medication rationing and restrictive household budgeting.

A pragmatic application of endobronchial ultrasound-guided transbronchial needle aspiration: a single institution experience.

BACKGROUND: Endobronchial ultrasound-guided trans-bronchial needle aspiration (EBUS-TBNA) is minimally invasive technique used for diagnosis and/or staging of benign and malignant pulmonary and non-pulmonary disease. Previous studies have established the utility of EBUS-TBNA in narrowly defined indications and populations. In this pragmatic 'real world' study we have analysed the use of EBUS-TBNA for a variety of clinical presentations and its clinical application in conjunction with other invasive investigations. METHODS: All EBUS-TBNA procedures performed at Sir Charles Gardiner Hospital in 2012-2014 were reviewed retrospectively, using relevant hospital databases. RESULTS: A total of 327 patients underwent 337 EBUS-TBNA procedures. EBUS-TBNA procedures were used to diagnose a wide spectrum of benign and malignant conditions. The main application was in the diagnosis and staging of malignant conditions (70.6%), and in the diagnosis of benign conditions such as sarcoidosis 40 (12.2%), and silicoanthracosis 17 (5.2%). EBUS-TBNA was sufficient to diagnose and stage the disease as a single stand-alone invasive procedure in 191 (59.2%) patients. EBUS-TBNA was the final invasive procedure undertaken in 283 (87.6%) patients. Only 13.3% of non small cell lung cancer (NSCLC) patients who had EBUS-TBNA as a first investigation required multiple procedures compared to 51.1% of all NSCLC patients undergoing EBUS-TBNA. Overall sensitivity, specificity, NPV and diagnostic accuracy for EBUS-TBNA were 89.7, 100, 85.1 and 89.9%, respectively and three minor complications (0.9%) occurred as a result of the procedure. CONCLUSIONS: EBUS-TBNA was undertaken for a wide variety of clinical conditions. Good diagnostic accuracy and safety profiles were demonstrated for the procedure, supporting its application as a first line investigation in the diagnosis and/or staging of a range of malignant and benign conditions. Our study was unique in its documentation of the use of EBUS-TBNA in a real-world setting in conjunction with other invasive modalities. EBUS-TBNA was utilised as a stand alone invasive procedure in more than half of the patients. Importantly, in NSCLC, when EBUS-TBNA was performed as primary diagnostic and staging investigation, less patients underwent subsequent invasive procedures.

How communication between cancer patients and their specialists affect the quality and cost of cancer care.

PURPOSE: Communication in cancer care is multidimensional and may affect patient treatment decision-making and quality of life. This study examined cancer patients' perceptions of the communication with their cancer specialists and explored its impact on the care they received and the financial burden they experienced. METHODS: Semi-structured telephone interviews were conducted with 20 rural and 20 outer metropolitan Western Australians diagnosed with breast, lung, prostate or colorectal cancer. Thematic analysis using a phenomenological approach was undertaken to derive key themes regarding the communication experiences of the participants. RESULTS: Four main themes emerged: information context, communication about treatment options and treatment providers, communication about costs of treatment and impact of communication on continuity of care. The quality of the communication experienced by participants was variable and in many cases sub-optimal. This affected their ability to undertake well-informed decisions regarding treatment and providers and led to substantial out-of-pocket expenses for several participants. Whilst participants differed in their information needs and expectations, most participants trusted clinicians' treatment recommendations. CONCLUSIONS: Our results raise concerns about the quality of communication cancer patients receive during treatment and the repercussions for their treatment decisions and out-of-pocket expenses. Clear treatment and cost communication could empower patients in choosing treatment and providers. However, these findings suggest patients must remain vigilant during consultations and discuss available treatment pathways and their financial dimension to avoid costly treatments or missing out on available financial aid.

Out-of-pocket expenses experienced by rural Western Australians diagnosed with cancer.

PURPOSE: Out-of-pocket expenses (OOPE) can have a significant impact on patients' experiences of cancer treatment. This cross-sectional study sought to quantify the OOPEs experienced by rural cancer patients in Western Australia (WA), and determine factors that contributed to higher OOPE. METHODS: Four hundred people diagnosed with breast, lung, colorectal or prostate cancer who resided in selected rural regions of WA were recruited through the WA Cancer Registry and contacted at least 3 months after diagnosis to report the medical OOPE (such as surgery or chemotherapy, supportive care, medication and tests) and non-medical OOPE (such as travel costs, new clothing and utilities) they had experienced as a result of accessing and receiving treatment. Bootstrapped t tests identified demographic, financial and treatment-related factors to include in multivariate analysis, performed using log-linked generalised linear models with gamma distribution. RESULTS: After a median 21 weeks post-diagnosis, participants experienced an average OOPE of AU$2179 (bootstrapped 95% confidence interval $1873-$2518), and 45 (11%) spent more than 10% of their household income on these expenses. Participants likely to experience higher total OOPE were younger than 65 years (p = 0.008), resided outside the South West region (p = 0.007) and had private health insurance (PHI) (p < 0.001). CONCLUSIONS: Rural WA cancer patients experience significant OOPE following their diagnosis. The impact these expenses have on patient wellbeing and their treatment decisions need to be further explored.

Implementing Endobronchial Ultrasound-Guided (EBUS) for Staging and Diagnosis of Lung Cancer: A Cost Analysis.

BACKGROUND Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and guide sheath (EBUS-GS) are gaining popularity for diagnosis and staging of lung cancer compared to CT-guided transthoracic needle aspiration (CT-TTNA), blind fiber-optic bronchoscopy, and mediastinoscopy. This paper aimed to examine predictors of higher costs for diagnosing and staging lung cancer, and to assess the effect of EBUS techniques on hospital cost. MATERIAL AND METHODS Hospital costs for diagnosis and staging of new primary lung cancer patients presenting in 2007-2008 and 2010-2011 were reviewed retrospectively. Multiple linear regression was used to determine relationships with hospital cost. RESULTS We reviewed 560 lung cancer patient records; 100 EBUS procedures were performed on 90 patients. Higher hospital costs were associated with: EBUS-TBNA performed (p<0.0001); increasing inpatient length of stay (p<0.0001); increasing number of other surgical/diagnostic procedures (p<0.0001); whether the date of management decision fell within an inpatient visit (p<0.0001); and if the patient did not have a CT-TTNA, then costs increased as the number of imaging events increased (interaction p<0.0001). Cohort was not significantly related to cost. Location of the procedure (outside vs. inside theater) was a predictor of lower one-day EBUS costs (p<0.0001). Cost modelling revealed potential cost saving of $1506 per EBUS patient if all EBUS procedures were performed outside rather than in the theater ($66,259 per annum). CONCLUSIONS EBUS-TBNA only was an independent predictor of higher cost for diagnosis and staging of lung cancer. Performing EBUS outside compared to in the theater may lower costs for one-day procedures; potential future savings are considerable if more EBUS procedures could be performed outside the operating theater.

Admissions for antibiotic-resistant infections in cancer patients during first year of cancer diagnosis: a cross-sectional study.

In this study, linked Western Australian health data were used to determine presence of an antibiotic-resistant infection (ABRI) for all people diagnosed with a primary invasive cancer in 2009. Of 10 858 cancer cases, 154 (1.42%) had an ABRI. Patients with an ABRI were older (71.5 vs 66 years), and more had died in the year following diagnosis (37.7 vs 20.2%, P < 0.001). The ABRI cohort had a higher proportion of colorectal, genitourinary and haematological cancers (19.5 vs 11.9%; 14.3 vs 9.7% and 16.9 vs 5.8%, respectively). Hospital admissions with an ABRI were longer (22.3 vs 2.9 days, P < 0.001) and had a higher proportion of unplanned admissions (60.3 vs 15.2%), admissions through emergency department (36.8 vs 8.3%) and intensive care admissions (14.9 vs 1.7%, P < 0.001). Patients with solid tumours who developed an ABRI were more likely to have received chemotherapy (35.9 vs 27.8%, P = 0.04). In haematological cancer patients, a greater proportion of the admissions with an ABRI occurred after radiation therapy or chemotherapy (P = 0.01 and P = 0.005, respectively). This study is the first to report population-level data on ABRI in cancer patients. Patients with an ABRI had more hospital admissions and poorer outcomes.

Development of a peer-review framework for cancer multidisciplinary meetings.

BACKGROUND: There is no mechanism in place for monitoring or quality improvement of cancer multidisciplinary meetings (MDM) in Australia. AIM: To develop a peer-review process for quality improvement of MDM. METHODS: This project involved three phases: (i) development of a draft peer-review framework, supporting documents and peer-review process; (ii) consultation with key stakeholders; (iii) refinement of the framework, documents and processes following a pilot study with three MDM. RESULTS: Feedback indicated that specific standards included in the framework needed to allow the peer reviewers to be flexible relative to the circumstances of the individual MDM. Conversely, feedback identified the need for clear, evidence-based clinical practice guidelines for the conduct of MDM, with accepted standards and objective measures of performance. MDM members were divided about the need to employ peer reviewers from the tumour stream of the MDM under review but agreed that closer involvement of the team under review to support the implementation of recommendations is warranted. CONCLUSIONS: We developed an adaptable peer-review framework and process using the current available evidence and guidance. While further research is needed to establish what constitutes best practice in MDM and which processes contribute to improved patient outcomes, the structured peer-review process we describe, when modified using the disease-relevant evidence, could be utilised more broadly as a quality improvement tool.

Csk-binding protein controls red blood cell development via regulation of Lyn tyrosine kinase activity.

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.

Impact of the introduction of EBUS on time to management decision, complications, and invasive modalities used to diagnose and stage lung cancer: a pragmatic pre-post study.

BACKGROUND: Utilisation of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and guide sheath (EBUS-GS) for diagnosis and staging of lung cancer is gaining popularity, however, its impact on clinical practice is unclear. This study aimed to determine the impact of the introduction of endobronchial ultrasound-guided procedures (EBUS) on time to management decision for lung cancer patients, and on the utilisation of other invasive diagnostic modalities, including CT-guided trans-thoracic needle aspiration (CT-TTNA), bronchoscopy, and mediastinoscopy. METHODS: Hospital records of new primary lung cancer patients presenting in 2007 and 2008 (Pre-EBUS cohort) and in 2010 and 2011 (Post-EBUS cohort) were reviewed retrospectively. RESULTS: The Pre-EBUS cohort included 234 patients. Of the 326 patients in the Post-EBUS cohort, 90 had an EBUS procedure (EBUS-TBNA for 19.0% and EBUS-GS for 10.4% of cases). The number of CT-TTNAs and bronchoscopies decreased following the introduction of EBUS (p = 0.015 and p < 0.001 respectively). Of 162 CT-TTNAs, 59 (36%) resulted in complications compared to 1 complication each for bronchoscopy and EBUS-GS, and no complications from EBUS-TBNA. Fewer complications occurred overall in the Post-EBUS cohort compared to the Pre-EBUS cohort (p = 0.0264). The median time to management decision was 17 days (IQR 24) for the Pre-EBUS and 13 days (IQR 21) for the Post-EBUS cohort (p = 0.07). Within the Post-EBUS cohort, median time to management decision was longer for the EBUS group (n = 90) than the Non-EBUS group (17 days (IQR 29) vs. 10 days (IQR 10), p < 0.001). For half of EBUS-TBNA patients (n = 28, 50.0%) and EBUS-GS patients (n = 14, 50.0 %), EBUS alone provided sufficient diagnostic and/or staging information; these patients had median time to management decision of 10 days. Regression analysis revealed that the number of imaging events, inpatient, and outpatient visits were significant predictors of time to management decision of >28 days; EBUS was not a predictor of time to management decision. CONCLUSIONS: The introduction of EBUS led to fewer CT-TTNAs and bronchoscopies and did not impact on the time to management decision. EBUS-TBNA or EBUS-GS alone provided sufficient information for diagnosis and/or regional staging in half of the lung cancer patients referred for this investigation.

Peer review of cancer multidisciplinary teams: is it acceptable in Australia?

OBJECTIVE: To develop a peer-review model for assessment and quality improvement of cancer multidisciplinary teams (MDTs) and qualitatively assess its feasibility and acceptability in Australia. DESIGN, SETTING AND PARTICIPANTS: A peer-review methodology was developed, based on the United Kingdom's National Health Service peer-review model and a comprehensive literature review. This was pilot tested in three mature MDTs in different settings. Semi-structured interviews were conducted between December 2012 and July 2013 with all five peer reviewers and 17 MDT members. Thematic analysis was undertaken using a framework approach. RESULTS: Peer reviewers and MDT members found the process reasonable, constructive and useful; however, those involved in the preparation for the review found it time-consuming. Most MDT members considered the final report accurate and reflective of their service. Recommendations in the report were met with mixed reactions: several MDT members perceived some recommendations to be particularly relevant, while others viewed the same recommendations as impractical or of limited value. Many participants were unsure if recommendations would be fully implemented. The majority saw value in the process and expressed support for its implementation locally and nationally; however, feedback suggests the most appropriate format is yet to be established. CONCLUSIONS: Peer review of cancer MDTs is feasible and acceptable. We describe valuable lessons learnt and recognise that further development of the proposed peer-review model and national benchmarking of MDTs against established outcome measures is required if this process is to be widely implemented.

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival.

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/ß (glycogen synthase kinase α/ß) phosphorylation and diminished p27(Kip1) 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.

Gain-of-function Lyn induces anemia: appropriate Lyn activity is essential for normal erythropoiesis and Epo receptor signaling.

Lyn is involved in erythropoietin (Epo)-receptor signaling and erythroid homeostasis. Downstream pathways influenced following Lyn activation and their significance to erythropoiesis remain unclear. To address this, we assessed a gain-of-function Lyn mutation (Lyn(up/up)) on erythropoiesis and Epo receptor signaling. Adult Lyn(up/up) mice were anemic, with dysmorphic red cells (spherocyte-like, acanthocytes) in their circulation, indicative of hemolytic anemia and resembling the human disorder chorea acanthocytosis. Heterozygous Lyn(+/up) mice became increasingly anemic with age, indicating that the mutation was dominant. In an attempt to overcome this anemia, extramedullary erythropoiesis was activated. As the mice aged, the levels of different immature erythroid populations changed, indicating compensatory mechanisms to produce more erythrocytes were dynamic. Changes in Epo signaling were observed in Lyn(+/up) erythroid cell lines and primary CD71(+) Lyn(up/up) erythroblasts, including significant alterations to the phosphorylation of Lyn, the Epo receptor, Janus kinase 2, Signal Transducer and Action of Transcription-5, GRB2-associated-binding protein-2, Akt, and Forkhead box O3. As a consequence of altered Lyn signaling, Lyn(+/up) cells remained viable in the absence of Epo but displayed delayed Epo-induced differentiation. These data demonstrate that Lyn gene dosage and activity are critical for normal erythropoiesis; constitutively active Lyn alters Epo signaling, which in turn produces erythroid defects.

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1.

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn.

Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria.

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.