Randy Wei

Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint

Hec1 contributes to accurate chromosome segregation by mediating spindle assembly checkpoint (SAC) signals and microtubule binding to ensure proper spindle assembly. We found that serine 165 of Hec1 was phosphorylated preferentially at kinetochores of misaligned chromosomes. This phosphorylation is important for SAC maintenance via Mad1/Mad2 localization to kinetochores.

Hice1, a Novel Microtubule-Associated Protein Required for Maintenance of Spindle Integrity and Chromosomal Stability in Human Cells

ABSTRACT Spindle integrity is critical for efficient mitotic progression and accurate chromosome segregation. Deregulation of spindles often leads to structural and functional aberrations, ultimately promoting segregation errors and aneuploidy, a hallmark of most human cancers. Here we report the characterization of a previously identified human sarcoma antigen (gene located at 19p13.11), Hice1, an evolutionarily nonconserved 46-kDa coiled-coil protein. Hice1 shows distinct cytoplasmic localization and associates with interphase centrosomes and mitotic spindles, preferentially at the spindle pole vicinity. Depletion of Hice1 by RNA interference resulted in abnormal and unstable spindle configurations, mitotic delay at prometaphase and metaphase, and elevated aneuploidy. Conversely, loss of Hice1 had minimal effects on interphase centrosome duplication. We also found that both full-length Hice1 and Hice1-N1, which is composed of 149 amino acids of the N-terminal region, but not the mutant lacking the N-terminal region, exhibited activities of microtubule bundling and stabilization at a near-physiological concentration. Consistently, overexpression of Hice1 rendered microtubule bundles in cells resistant to nocodazole- or cold-treatment-induced depolymerization. These results demonstrate that Hice1 is a novel microtubule-associated protein important for maintaining spindle integrity and chromosomal stability, in part by virtue of its ability to bind, bundle, and stabilize microtubules.

Mutation of NIMA-related kinase 1 (NEK1) leads to chromosome instability

BackgroundNEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (NIMA), is involved early in the DNA damage sensing/repair pathway. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks after low dose ionizing radiation (IR). NEK1-deficient cells also fail to activate the checkpoint kinases CHK1 and CHK2, and fail to arrest properly at G1/S or G2/M-phase checkpoints after DNA damage.ResultsWe show here that NEK1-deficient cells suffer major errors in mitotic chromosome segregation and cytokinesis, and become aneuploid. These NEK1-deficient cells transform, acquire the ability to grow in anchorage-independent conditions, and form tumors when injected into syngeneic mice. Genomic instability is also manifest in NEK1 +/- mice, which late in life develop lymphomas with a much higher incidence than wild type littermates.ConclusionNEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.

Bone mineral density loss in thoracic and lumbar vertebrae following radiation for abdominal cancers.

PURPOSE To investigate the relationship between abdominal chemoradiation (CRT) for locally advanced cancers and bone mineral density (BMD) reduction in the vertebral spine. MATERIALS AND METHODS Data from 272 patients who underwent abdominal radiation therapy from January 1997 to May 2015 were retrospectively reviewed. Forty-two patients received computed tomography (CT) scans of the abdomen prior to initiation and at least twice after radiation therapy. Bone attenuation (in Hounsfield unit) (HU) measurements were collected for each vertebral level from T7 to L5 using sagittal CT images. Radiation point dose was obtained at each mid-vertebral body from the radiation treatment plan. Percent change in bone attenuation (Δ%HU) between baseline and post-radiation therapy were computed for each vertebral body. The Δ%HU was compared against radiation dose using Pearsons linear correlation. RESULTS Abdominal radiotherapy caused significant reduction in vertebral BMD as measured by HU. Patients who received only chemotherapy did not show changes in their BMD in this study. The Δ%HU was significantly correlated with the radiation point dose to the vertebral body (R=-0.472, P<0.001) within 4-8 months following RT. The same relationship persisted in subsequent follow up scans 9 months following RT (R=-0.578, P<0.001). Based on the result of linear regression, 5 Gy, 15 Gy, 25 Gy, 35 Gy, and 45 Gy caused 21.7%, 31.1%, 40.5%, 49.9%, and 59.3% decrease in HU following RT, respectively. Our generalized linear model showed that pre-RT HU had a positive effect (β=0.830) on determining post-RT HU, while number of months post RT (β=-0.213) and radiation point dose (β=-1.475) had a negative effect. A comparison of the predicted versus actual HU showed significant correlation (R=0.883, P<0.001) with the slope of the best linear fit=0.81. Our models predicted HU were within ±20 HU of the actual value in 53% of cases, 70% of the predictions were within ±30 HU, 81% were within ±40 HU, and 90% were within ±50 HU of the actual post-RT HU. Four of 42 patients were found to have vertebral body compression fractures in the field of radiation. CONCLUSIONS Patients who receive abdominal chemoradiation develop significant BMD loss in the thoracic and lumbar vertebrae. Treatment-related BMD loss may contribute to the development of vertebral compression fractures. A predictive model for post-CRT BMD changes may inform bone protective strategies in patients planned for abdominal CRT.

Hec1 Contributes to Mitotic Centrosomal Microtubule Growth for Proper Spindle Assembly through Interaction with Hice1

Previous studies have stipulated Hec1 as a conserved kinetochore component critical for mitotic control in part by directly binding to kinetochore fibers of the mitotic spindle and by recruiting spindle assembly checkpoint proteins Mad1 and Mad2. Hec1 has also been reported to localize to centrosomes, but its function there has yet to be elucidated. Here, we show that Hec1 specifically colocalizes with Hice1, a previously characterized centrosomal microtubule-binding protein, at the spindle pole region during mitosis. In addition, the C-terminal region of Hec1 directly binds to the coiled-coil domain 1 of Hice1. Depletion of Hice1 by small interfering RNA (siRNA) reduced levels of Hec1 in the cell, preferentially at centrosomes and spindle pole vicinity. Reduction of de novo microtubule nucleation from mitotic centrosomes can be observed in cells treated with Hec1 or Hice1 siRNA. Consistently, neutralization of Hec1 or Hice1 by specific antibodies impaired microtubule aster formation from purified mitotic centrosomes in vitro. Last, disruption of the Hec1/Hice1 interaction by overexpressing Hice1DeltaCoil1, a mutant defective in Hec1 interaction, elicited abnormal spindle morphology often detected in Hec1 and Hice1 deficient cells. Together, the results suggest that Hec1, through cooperation with Hice1, contributes to centrosome-directed microtubule growth to facilitate establishing a proper mitotic spindle.

Attitudes of radiation oncologists toward palliative and supportive care in the United States: Report on national membership survey by the American Society for Radiation Oncology (ASTRO)

PURPOSE Radiation oncologists are frequently involved in providing palliative and supportive care (PSC) for patients with advanced cancers through delivery of palliative radiation. Whether they are confident in their ability to assess and initiate treatments for pain, nonpain, and psychosocial distress is unknown. The American Society for Radiation Oncology surveyed its practicing members in the United States on self-assessment of their primary PSC skills and access to continuing medical education on PSC. METHODS We electronically surveyed 4093 practicing radiation oncologists in the United States. The survey consisted of 16-questions in 5 sections1: demographics,2 PSC training,3 domains of PSC,4 perceived barriers as a radiation oncologist to initiate advanced care planning, and5 discussion of prognosis. RESULTS The survey was e-mailed to 4093 American Society for Radiation Oncology members, and 649 responses were received (response rate 16%). The majority (91%) of radiation oncologists surveyed believe PSC is an important competency for radiation oncologists. Most radiation oncologists reported that they are moderately confident in their ability to assess and manage pain and gastrointestinal symptoms, but less confident in their ability to manage anorexia, anxiety, and depression. Despite areas of decreased confidence, a large number (42%) of radiation oncologists do not receive any additional PSC education beyond their residency training. Lastly, a perceived fear of upsetting referring medical oncologists and lack of clinic time are concerns for radiation oncologists who may want to initiate goals of care/advance care planning discussions with patients and their families. CONCLUSION Radiation oncologists are more confident in their ability to assess and manage pain than in their ability to manage depression, anxiety, anorexia, and fatigue. There is a need for increasing continuing medical educational efforts in PSC for practicing radiation oncologists, and strengthening PSC training in residency programs.

The dosimetric impact of image guided radiation therapy by intratumoral fiducial markers

PURPOSE Pancreatic fiducials have proven superior over other isocenter localization surrogates, including anatomical landmarks and intratumoral or adjacent stents. The more clinically relevant dosimetric impact of image guided radiation therapy (IGRT) using intratumoral fiducial markers versus bony anatomy has not yet been described and is therefore the focus of the current study. METHODS AND MATERIALS Using daily orthogonal kV or cone beam computed tomography (CBCT) images and positional and dosimetric data were analyzed for 12 consecutive patients treated with fiducial based IGRT and volumetric modulated arc therapy to the intact pancreas. The shifts from fiducial to bone (ΔFid-Bone) required to realign the daily fiducial-matched pretreatment images (kV, CBCTs) to the planning computed tomography (CT) using bony anatomic landmarks were recorded. The isocenter was then shifted by (ΔFid-Bone) for 5 evenly spaced treatments, and the dosimetric impact of ΔFid-Bone was calculated for planning target volume coverage (PTV50.4 and PTV47.9) and organs at risk (liver, kidney, and stomach/duodenum). RESULTS The ΔFid-Bone were greatest in the superoinferior direction (ΔFid-Bone anteroposterior, 2.7 ± 3.0; left-right, 2.8 ± 2.8; superoinferior, 6.3 ± 7.9 mm; mean ± standard deviation; P = .03). PTV50.4 coverage was reduced by 13% (fiducial plan 95 ± 2.0 vs bone plan 82 ± 12%; P = .005; range, 5%-52%; >5% loss in all; and >10% loss in 42% of patients), and to a lesser degree for PTV47.9 (difference, -8%; range, 1%-30%; fiducial plan 100 ± 0.3% vs bone plan 92 ± 7.6%; P = .003; with reductions of >5% in 66% and >10% in 33% of patients). The dosimetric impact of ΔFid-Bone on the organs at risk was not significant. Positional shifts for kV- and CBCT-based realignments were nearly identical. CONCLUSION Compared with matching by fiducial markers, IGRT matched by bony anatomy substantially reduces the PTV50.4 and PTV47.9 coverage, supporting the use of intratumoral pancreatic markers for improved targeting in IGRT for pancreatic cancer.

Complementary interhelical interactions between three buried Glu-Lys pairs within three heptad repeats are essential for Hec1-Nuf2 heterodimerization and mitotic progression.

Background: Hec1 and Nuf2 are components of the NDC80 complex essential for faithful chromosome segregation. Results: Three contiguous heptad repeats with buried interhelical Glu-Lys pairs are required for Hec1-Nuf2 dimerization, NDC80 complex formation, and mitotic progression. Conclusion: Interhelical electrostatic and hydrophobic interactions define specificity and stability requirements for Hec1-Nuf2 dimerization. Significance: The results elucidate how Hec1-Nuf2 dimerize and provide insight into NDC80 complex formation. Hec1 and Nuf2, core components of the NDC80 complex, are essential for kinetochore-microtubule attachment and chromosome segregation. It has been shown that both Hec1 and Nuf2 utilize their coiled-coil domains to form a functional dimer; however, details of the consequential significance and structural requirements to form the dimerization interface have yet to be elucidated. Here, we showed that Hec1 required three contiguous heptad repeats from Leu-324 to Leu-352, but not the entire first coiled-coil domain, to ensure overall stability of the NDC80 complex through direct interaction with Nuf2. Substituting the hydrophobic core residues, Leu-331, Val-338, and Ile-345, of Hec1 with alanine completely eliminated Nuf2 binding and blocked mitotic progression. Moreover, unlike most coiled-coil proteins, where the buried positions are composed of hydrophobic residues, Hec1 possessed an unusual distribution of glutamic acid residues, Glu-334, Glu-341, and Glu-348, buried within the interior dimerization interface, which complement with three Nuf2 lysine residues: Lys-227, Lys-234, and Lys-241. Substituting these corresponding residues with alanine diminished the binding affinity between Hec1 and Nuf2, compromised NDC80 complex formation, and adversely affected mitotic progression. Taken together, these findings demonstrated that three buried glutamic acid-lysine pairs, in concert with hydrophobic interactions of core residues, provide the major specificity and stability requirements for Hec1-Nuf2 dimerization and NDC80 complex formation.

Palliative care and palliative radiation therapy education in radiation oncology: A survey of US radiation oncology program directors

PURPOSE The purpose of this study was to assess the state of palliative and supportive care (PSC) and palliative radiation therapy (RT) educational curricula in radiation oncology residency programs in the United States. METHODS AND MATERIALS We surveyed 87 program directors of radiation oncology residency programs in the United States between September 2015 and November 2015. An electronic survey on PSC and palliative RT education during residency was sent to all program directors. The survey consisted of questions on (1) perceived relevance of PSC and palliative RT to radiation oncology training, (2) formal didactic sessions on domains of PSC and palliative RT, (3) effective teaching formats for PSC and palliative RT education, and (4) perceived barriers for integrating PSC and palliative RT into the residency curriculum. RESULTS A total of 57 responses (63%) was received. Most program directors agreed or strongly agreed that PSC (93%) and palliative radiation therapy (99%) are important competencies for radiation oncology residents and fellows; however, only 67% of residency programs had formal educational activities in principles and practice of PSC. Most programs had 1 or more hours of formal didactics on management of pain (67%), management of neuropathic pain (65%), and management of nausea and vomiting (63%); however, only 35%, 33%, and 30% had dedicated lectures on initial management of fatigue, assessing role of spirituality, and discussing advance care directives, respectively. Last, 85% of programs reported having a formal curriculum on palliative RT. Programs were most likely to have education on palliative radiation to brain, bone, and spine, but less likely on visceral, or skin, metastasis. CONCLUSIONS Residency program directors believe that PSC and palliative RT are important competencies for their trainees and support increasing education in these 2 educational domains. Many residency programs have structured curricula on PSC and palliative radiation education, but room for improvement exists in management of fatigue, assessing role of spirituality, and discussion regarding advance care planning.

Radiation dermatitis caused by a bolus effect from an abdominal compression device.

American Association of Physicists in Medicine (AAPM) Task Group 176 evaluated the dosimetric effects caused by couch tops and immobilization devices. The report analyzed the extensive physics-based literature on couch tops, stereotactic body radiation therapy (SBRT) frames, and body immobilization bags, while noting the scarcity of clinical reports of skin toxicity because of external devices. Here, we present a clinical case report of grade 1 abdominal skin toxicity owing to an abdominal compression device. We discuss the dosimetric implications of the utilized treatment plan as well as post hoc alternative plans and quantify differences in attenuation and skin dose/build-up between the device, a lower-density alternative device, and an open field. The description of the case includes a 66-year-old male with HER2 amplified poorly differentiated distal esophageal adenocarcinoma treated with neoadjuvant chemo-radiation and the use of an abdominal compression device. Radiation was delivered using volumetric modulated arc therapy (VMAT) with 2 arcs using abdominal compression and image guidance. The total dose was 50.4Gy delivered over 40 elapsed days. With 2 fractions remaining, the patient developed dermatitis in the area of the compression device. The original treatment plan did not include a contour of the device. Alternative post hoc treatment plans were generated, one to contour the device and a second with anterior avoidance. In conclusion, replanning with the device contoured revealed the bolus effect. The skin dose increased from 27 to 36Gy. planned target volume (PTV) coverage at 45Gy was reduced to 76.5% from 95.8%. The second VMAT treatment plan with an anterior avoidance sector and more oblique beam angles maintained PTV coverage and spared the anterior wall, however at the expense of substantially increased dose to lung. This case report provides an important reminder of the bolus effect from external devices such as abdominal compression. Special consideration must be given to contour and/or avoiding beam entrance to the device, and to the use of such devices in patients who may have heightened radiosensitivity, such as those who are human immunodeficiency virus (HIV)-positive.