Combination of dasatinib and venetoclax in newly diagnosed chronic phase chronic myeloid leukemia.

BACKGROUND: The dual inhibition of the BCR::ABL1 tyrosine kinase and BCL-2 could potentially deepen the response rates of chronic myeloid leukemia in chronic phase (CML-CP). This study evaluated the safety and efficacy of the combination of dasatinib and venetoclax. METHODS: In this phase 2 trial, patients with CML-CP or accelerated phase (clonal evolution) received dasatinib 50 mg/day for three courses; venetoclax was added in course 4 for 3 years. The initial venetoclax dose was 200 mg/day continuously but reduced later to 200 mg/day for 14 days, and to 100 mg/day for 7 days per course once a molecular response (MR)4.5 was achieved. After 3 years of combination, patients were maintained on single-agent dasatinib. The primary end point was the rate of major molecular response (MMR) by 12 months of combination. RESULTS: Sixty-five patients were treated. Their median age was 46 years (range, 23-73). By 12 months of combination, the MMR, MR4, and MR4.5 rates were 86%, 53%, and 45%, respectively. After a median follow-up of 42 months, the 4-year event-free and overall survival rates were 96% and 100%, respectively. Outcomes with the combination were comparable to historical outcomes with single-agent dasatinib (cumulative 12-months MMR rate of 79% with both strategies). The incidence of grade 3-4 neutropenia was 22% with the combination and 11% with single-agent dasatinib (p < .001). CONCLUSIONS: Treatment with dasatinib and venetoclax was safe and effective in CML-CP. The cumulative response rates with the combination were similar to those with single-agent dasatinib. Further follow-up is needed to evaluate the rates of durable deep molecular response and treatment-free remission.

Low-dose dasatinib 50 mg/day versus standard-dose dasatinib 100 mg/day as frontline therapy in chronic myeloid leukemia in chronic phase: A propensity score analysis.

Low-dose dasatinib is safe and effective in patients with chronic myeloid leukemia in chronic phase (CML-CP). No randomized trials have compared the outcome with standard-dose dasatinib. This study aims to compare the outcome of patients with CML-CP treated with frontline dasatinib 50 versus 100 mg/day. We analyzed 233 patients with newly diagnosed CML-CP treated with low-dose dasatinib (N = 83) or standard-dose dasatinib (N = 150). Propensity score analysis with 1:1 matching was performed and identified 77 patients in each cohort without significant baseline differences. Response rates were reported as the cumulative incidences of complete cytogenetic response, major molecular response (MMR), molecular response (MR)4, and MR4.5. Failure-free survival (FFS), event-free survival (EFS), transformation-free survival (TFS), and overall survival (OS) were also compared. Patients on low-dose dasatinib with suboptimal response by European LeukemiaNet (ELN) 2013 criteria had the option to increase the dose to 100 mg/day. The overall median follow-up time was 60 months. The 3-year MMR rates were 92% and 84% for low-dose and standard-dose dasatinib, respectively (p = .23). Dasatinib 50 mg/day induced higher cumulative incidence of MR4 (77% vs. 66%; p = .04) and MR4.5 (77% vs. 62%; p = .02) at 3 years. The 4-year FFS, EFS and OS rates were 89% versus 77% (p = .04), 95% versus 92% (p = .06), and 97% versus 96% (p = .78) with low-dose and standard-dose dasatinib, respectively. The rate of any grade pleural effusion was 5% with dasatinib 50 mg/day compared to 21% with 100 mg/day. Dasatinib 50 mg/day is at least as effective as 100 mg/day with a better safety profile and drug exposure.

The LEukemia Artificial Intelligence Program (LEAP) in chronic myeloid leukemia in chronic phase: A model to improve patient outcomes.

Extreme gradient boosting methods outperform conventional machine-learning models. Here, we have developed the LEukemia Artificial intelligence Program (LEAP) with the extreme gradient boosting decision tree method for the optimal treatment recommendation of tyrosine kinase inhibitors (TKIs) in patients with chronic myeloid leukemia in chronic phase (CML-CP). A cohort of CML-CP patients was randomly divided into training/validation (N = 504) and test cohorts (N = 126). The training/validation cohort was used for 3-fold cross validation to develop the LEAP CML-CP model using 101 variables at diagnosis. The test cohort was then applied to the LEAP CML-CP model and an optimum TKI treatment was suggested for each patient. The area under the curve in the test cohort was 0.81899.Backward multivariate analysis identified age at diagnosis, the degree of comorbidities, and TKI recommended therapy by the LEAP CML-CP model as independent prognostic factors for overall survival. The bootstrapping method internally validated the association of the LEAP CML-CP recommendation with overall survival as an independent prognostic for overall survival. Selecting treatment according to the LEAP CML-CP personalized recommendations, in this model, is associated with better survival probability compared to treatment with a LEAP CML-CP non-recommended therapy. This approach may pave a way of new era of personalized treatment recommendations for patients with cancer.

Long-term results of frontline dasatinib in chronic myeloid leukemia.

BACKGROUND: Dasatinib is a second-generation tyrosine kinase inhibitor that, when used as frontline therapy, produces more and faster cytogenetic and molecular responses compared with imatinib. The authors report the long-term follow-up from the first study using dasatinib as initial therapy for chronic-phase chronic myeloid leukemia. METHODS: Between November 2005 and August 2014, patients were randomly assigned to receive 100 mg daily or 50 mg twice daily. After June 2009, all patients started with 100 mg daily. RESULTS: With a median follow-up of 6.5 years, 94 of 149 treated patients (63%) were still receiving dasatinib on study. The median patient age was 48 years (interquartile range, 37-55 years), and 9% of patients had a high risk Sokal risk score. The cumulative complete cytogenetic response rate at 11 years was 92.6%, the major molecular response (MR) rate was 88.2%, and the MR4.5 rate (indicating a ≥4.5-log reduction in BCR-ABL1 transcripts) was 79.5%. The median time to a major MR and MR4.5 was 6 and 23 months, respectively. A sustained MR4.5 (≥2 years) was achieved in 82 patients (55%). The 10-year overall survival, transformation-free survival, event-free survival, and failure-free survival rates were 89%, 95%, 86%, and 65%, respectively. Univariate analysis showed that the achievement of a complete MR was associated with improved overall survival. The most common reasons for treatment discontinuation were toxicity and elective discontinuation. The most common treatment-emergent grade 3 and 4 adverse events were fatigue, thrombocytopenia, and infections. CONCLUSIONS: After this long-term follow-up, dasatinib continues to show an excellent safety profile and produces rapid cytogenetic responses and MRs, durable deep MRs, and excellent long-term survival outcomes in patients with chronic-phase chronic myeloid leukemia.

Long-term follow-up of lower dose dasatinib (50 mg daily) as frontline therapy in newly diagnosed chronic-phase chronic myeloid leukemia.

BACKGROUND: Dasatinib, a potent Bcr-Abl tyrosine kinase inhibitor, is approved for the treatment of chronic-phase chronic myeloid leukemia (CML-CP) in the frontline and salvage settings. Notable side effects include pleural effusions and myelosuppression. Dasatinib at 50 mg daily has previously been reported to be active and better tolerated than the approved 100-mg daily dose. The aim of this study was to update the long-term follow-up results of dasatinib at 50 mg daily as frontline therapy for CML-CP. METHODS: Eighty-three patients with newly diagnosed CML-CP received dasatinib at 50 mg daily. Eligibility and response criteria were standards used in previous protocols. RESULTS: After a minimum follow-up of 12 months, 81 patients were evaluable. Two patients came off the study in less than 3 months. The rates of BCR-ABL1 transcript levels (International Standard) at ≤10% and ≤1% at 3 months were 96% and 77%, respectively. The cumulative rates for a complete cytogenetic response by 6 and 12 months were 77% and 95%, respectively. The cumulative rates for a major molecular response, a molecular response with a 4.0-log reduction, and a molecular response with a 4.5-log reduction by 12 months were 81%, 55%, and 49%, respectively. Twenty-one patients (25%) had treatment interruptions for a median of 13 days (range, 4-64 days). Five patients (6%) developed pleural effusions; 4 of these patients (80%) required a dose reduction. Two patients (2%) failed to achieve any cytogenetic or molecular response and were taken off the study. At a median follow-up of 24 months, none of the patients had disease transformation to an accelerated or blastic phase. The 2-year event-free and overall survival rates were 100%. CONCLUSIONS: These updated results continue to support 50 mg of dasatinib daily as an effective and safe dose for early CML-CP.

Early results of lower dose dasatinib (50 mg daily) as frontline therapy for newly diagnosed chronic-phase chronic myeloid leukemia.

BACKGROUND: Dasatinib is a potent BCR-ABL1 and Src family tyrosine kinase inhibitor. It is approved at a dose of 100 mg orally daily for the treatment of chronic myeloid leukemia in chronic phase (CML-CP). This dose schedule is associated with myelosuppression and pleural effusions. Anecdotal data suggest that lower doses may be as effective and less toxic. The aim of this study was to assess the efficacy and safety of a lower dose of dasatinib (50 mg daily) in patients with newly diagnosed CML-CP. METHODS: Seventy-five patients with newly diagnosed CML-CP received dasatinib 50 mg daily. The eligibility and response criteria were standards used in previous protocols. RESULTS: At a median follow-up of 9 months, 60 patients were evaluable for a response at 3 months. The rates of patients achieving BCR-ABL1 transcript levels ≤ 10% and ≤ 1% at 3 months by the International Standard were 93% and 72%, respectively. The rates of complete cytogenetic response by conventional cytogenetics or fluorescence in situ hybridization at 6 and 12 months were 86% and 88%, respectively. At 12 months, 79%, 71%, and 46% of the patients had achieved a major molecular response, a molecular response with a 4.0-log reduction, and a molecular response with a 4.5-log reduction, respectively. Nine patients had a dose interruption for ≤14 days. Only 1 patient developed a pleural effusion requiring a dose reduction to 20 mg. All patients remained alive and with no transformation so far. CONCLUSION: Dasatinib 50 mg daily is active and well tolerated in patients with newly diagnosed CML-CP. It should be further explored as a new potential standard-of-care option for chronic myeloid leukemia. Cancer 2018;124:2740-2747. © 2018 American Cancer Society.

Expansion of an atypical NK cell subset in mouse models of systemic lupus erythematosus.

Chronic inflammatory conditions, such as in autoimmune disease, can disturb immune cell homeostasis and induce the expansion of normally rare cell populations. In our analysis of various murine models of lupus, we detect increased frequency of an uncommon subset identified as NK1.1(+)CD11c(+)CD122(+)MHC class II(+). These cells share characteristics with the NK cell lineage and with cells previously described as IFN-producing killer dendritic cells: 1) they depend on IL-15 and express E4BP4; 2) they are cytotoxic and produce type I and type II IFN upon activation; and 3) they are efficient APCs both through MHC class II expression and in cross-presentation to CD8s. These atypical NK cells are responsive to TLR stimulation and thus are most abundant in mice with high copy number of the Tlr7 gene. They are highly proliferative as assessed by in vivo BrdU incorporation. In adoptive transfer experiments they persist in high numbers for months and maintain their surface marker profile, indicating that this population is developmentally stable. Gene expression analyses on both mRNA and microRNAs show a modified cell cycle program in which various miR-15/16 family members are upregulated, presumably as a consequence of the proliferative signal mediated by the increased level of growth factors, Ras and E2F activity. Alternatively, low expression of miR-150, miR-181, and miR-744 in these cells implies a reduction in their differentiation capacity. These results suggest that cells of the NK lineage that undergo TLR stimulation might turn on a proliferative program in detriment of their full differentiation into mature NK cells.

Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation.

C-Repeat Binding Factors (CBFs) are DNA-binding transcriptional activators of gene pathways imparting freezing tolerance. Poaceae contain three CBF subfamilies, two of which, HvCBF3/CBFIII and HvCBF4/CBFIV, are unique to this taxon. To gain mechanistic insight into HvCBF4/CBFIV CBFs we overexpressed Hv-CBF2A in spring barley (Hordeum vulgare) cultivar 'Golden Promise'. The Hv-CBF2A overexpressing lines exhibited stunted growth, poor yield, and greater freezing tolerance compared to non-transformed 'Golden Promise'. Differences in freezing tolerance were apparent only upon cold acclimation. During cold acclimation freezing tolerance of the Hv-CBF2A overexpressing lines increased more rapidly than that of 'Golden Promise' and paralleled the freezing tolerance of the winter hardy barley 'Dicktoo'. Transcript levels of candidate CBF target genes, COR14B and DHN5 were increased in the overexpressor lines at warm temperatures, and at cold temperatures they accumulated to much higher levels in the Hv-CBF2A overexpressors than in 'Golden Promise'. Hv-CBF2A overexpression also increased transcript levels of other CBF genes at FROST RESISTANCE-H2-H2 (FR-H2) possessing CRT/DRE sites in their upstream regions, the most notable of which was CBF12. CBF12 transcript levels exhibited a relatively constant incremental increase above levels in 'Golden Promise' both at warm and cold. These data indicate that Hv-CBF2A activates target genes at warm temperatures and that transcript accumulation for some of these targets is greatly enhanced by cold temperatures.

Low-Dose Dasatinib (50 mg Daily) Frontline Therapy in Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia: 5-Year Follow-Up Results.

BACKGROUND: Dasatinib is a BCR::ABL1 tyrosine kinase inhibitor approved as frontline therapy at a 100 mg daily for chronic myeloid leukemia in chronic phase (CML-CP). The use of a lower dose of dasatinib (50 mg daily) has demonstrated better tolerance and improved outcomes compared with the standard dose. Here, we report the updated results in a large cohort with a 5-year follow-up. PATIENTS AND METHODS: Patients with newly diagnosed CML-CP were eligible. Entry and response-outcome criteria were standard. Dasatinib was given as 50 mg orally daily. RESULTS: Eighty-three patients were included. At 3 months, 78 (96%) patients achieved BCR::ABL1 transcripts (IS) ≤10%, and at 12 months, 65 (81%) patients achieved BCR::ABL1 transcript (IS) ≤0.1%. The cumulative incidence of complete cytogenetic, major molecular, and deep molecular responses at 5 years were 98%, 95%, and 82%, respectively. Rates of failures due to resistance (n = 4; 5%) and toxicity (n = 4; 5%) were low. The 5-year overall survival was 96% and event-free survival 90%. No transformations to accelerated or blastic phase were observed. Grade 3 to 4 pleural effusions developed in 2% of patients. CONCLUSION: Dasatinib 50 mg daily is an effective and safe treatment for newly diagnosed CML-CP.

A Clinical Nurse Specialist-Led Emergency Department Naloxone Distribution Program.

PURPOSE: This article describes the implementation of a clinical nurse specialist-led emergency department overdose education and naloxone distribution program. The program's purpose is to increase naloxone availability to reduce opiate overdose mortality rates within the local community. DESCRIPTION OF PROGRAM: The program distributes naloxone kits to patients in the emergency department after an opioid overdose. The kits are designed to help recipients prevent, recognize, and respond to an opioid overdose. OUTCOME: The program, which includes naloxone take-home kits and clinical guidelines outlining a standard of care for naloxone distribution and coprescribing, was successfully implemented across 11 emergency departments within an integrated health system. More than 250 kits were dispensed within the first year of program implementation along with an online patient education video that received more than 1600 views. In 2017, the county reported an opioid-related overdose death rate of 16.5 (per 100 000 residents). From January 2018 to June 2019, the opioid-related death rate per 100 000 residents was reported at 9.6. CONCLUSION: Although emergency department naloxone distribution programs are feasible in the acute care setting, it was critical for clinical nurse specialists to enlist an interdisciplinary team and engage executive leadership to ensure program success. For others considering such a program, early consideration should be given to determining financial support and evaluating the compliance and regulatory aspects of dispensing medications from emergency settings.

Ectopic AtCBF1 over-expression enhances freezing tolerance and induces cold acclimation-associated physiological modifications in potato.

We studied the effect of ectopic AtCBF over-expression on physiological alterations that occur during cold exposure in frost-sensitive Solanum tuberosum and frost-tolerant Solanum commersonii. Relative to wild-type plants, ectopic AtCBF1 over-expression induced expression of COR genes without a cold stimulus in both species, and imparted a significant freezing tolerance gain in both species: 2 degrees C in S. tuberosum and up to 4 degrees C in S. commersonii. Transgenic S. commersonii displayed improved cold acclimation potential, whereas transgenic S. tuberosum was still incapable of cold acclimation. During cold treatment, leaves of wild-type S. commersonii showed significant thickening resulting from palisade cell lengthening and intercellular space enlargement, whereas those of S. tuberosum did not. Ectopic AtCBF1 activity induced these same leaf alterations in the absence of cold in both species. In transgenic S. commersonii, AtCBF1 activity also mimicked cold treatment by increasing proline and total sugar contents in the absence of cold. Relative to wild type, transgenic S. commersonii leaves were darker green, had higher chlorophyll and lower anthocyanin levels, greater stomatal numbers, and displayed greater photosynthetic capacity, suggesting higher productivity potential. These results suggest an endogenous CBFpathway is involved in many of the structural, biochemical and physiological alterations associated with cold acclimation in these Solanum species.

Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield.

Solanum tuberosum is a frost-sensitive species incapable of cold acclimation. A brief exposure to frost can significantly reduce its yields, while hard frosts can completely destroy entire crops. Thus, gains in freezing tolerance of even a few degrees would be of considerable benefit relative to frost damage. The S. tuberosum cv. Umatilla was transformed with three Arabidopsis CBF genes (AtCBF1-3) driven by either a constitutive CaMV35S or a stress-inducible Arabidopsis rd29A promoter. AtCBF1 and AtCBF3 over-expression via the 35S promoter increased freezing tolerance about 2 degrees C, whereas AtCBF2 over-expression failed to increase freezing tolerance. Transgenic plants of AtCBF1 and AtCBF3 driven by the rd29A promoter reached the same level of freezing tolerance as the 35S versions within a few hours of exposure to low but non-freezing temperatures. Constitutive expression of AtCBF genes was associated with negative phenotypes, including smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production. While imparting the same degree of freezing tolerance, control of AtCBF expression via the stress-inducible promoter ameliorated these negative phenotypic effects and restored tuber production to levels similar to wild-type plants. These results suggest that use of a stress-inducible promoter to direct CBF transgene expression can yield significant gains in freezing tolerance without negatively impacting agronomically important traits in potato.

Transgenic sterility in Populus: expression properties of the poplar PTLF, Agrobacterium NOS and two minimal 35S promoters in vegetative tissues.

Transgenic sterility is a desirable trait for containment of many kinds of transgenes and exotic species. Genetically engineered floral sterility can be imparted by expression of a cytotoxin under the control of a predominantly floral-tissue-specific promoter. However, many otherwise desirable floral promoters impart substantial non-floral expression, which can impair plant health or make it impossible to regenerate transgenic plants. We are therefore developing a floral sterility system that is capable of attenuating undesired background vegetative expression. As a first step towards this goal, we compared the vegetative expression properties of the promoter of the poplar (Populus trichocarpa Torr. & Gray) homolog of the floral homeotic gene LEAFY (PTLF), which could be used to impart male and female flower sterility, to that of three candidate attenuator-gene promoters: the cauliflower mosaic virus (CaMV) 35S basal promoter, the CaMV 35S basal promoter fused to the TMV omega element and the nopaline synthase (NOS) promoter. The promoters were evaluated via promoter::GUS gene fusions in a transgenic poplar hybrid (Populus tremula L. x P. alba L.) by both histochemical and fluorometric GUS assays. In leaves, the NOS promoter conveyed the highest activity and had a mean expression level 5-fold higher than PTLF, whereas the CaMV 35S basal promoter fused to the omega element and the CaMV 35S basal promoter alone directed mean expression levels that were 0.5x and 0.35x that of PTLF, respectively. Differential expression in shoots, leaves, stems and roots was observed only for the NOS and PTLF promoters. Strongest expression was observed in roots for the NOS promoter, whereas the PTLF promoter directed highest expression in shoots. The NOS promoter appears best suited to counteract vegetative expression of a cytotoxin driven by the PTLF promoter where 1:1 toxin:attenuator expression is required.

Efficient and stable transgene suppression via RNAi in field-grown poplars.

The efficiency and stability of RNA interference (RNAi) in perennial species, particularly in natural environments, is poorly understood. We studied 56 independent poplar RNAi transgenic events in the field over 2 years. A resident BAR transgene was targeted with two different types of RNAi constructs: a 475-bp IR of the promoter sequence and a 275-bp IR of the coding sequence, each with and without the presence of flanking matrix attachment regions (MARs). RNAi directed at the coding sequence was a strong inducer of gene silencing; 80% of the transgenic events showed more than 90% suppression. In contrast, RNAi targeting the promoter resulted in only 6% of transgenic events showing more than 90% suppression. The degree of suppression varied widely but was highly stable in each event over 2 years in the field, and had no association with insert copy number or the presence of MARs. RNAi remained stable during a winter to summer seasonal cycle, a time when expression of the targeted transgene driven by an rbcS promoter varied widely. When strong gene suppression was induced by an IR directed at the promoter sequence, it was accompanied by methylation of the homologous promoter region. DNA methylation was also observed in the coding region of highly suppressed events containing an IR directed at the coding sequence; however, the methylation degree and pattern varied widely among those suppressed events. Our results suggest that RNAi can be highly effective for functional genomics and biotechnology of perennial plants.

Expression levels of barley Cbf genes at the Frost resistance-H2 locus are dependent upon alleles at Fr-H1 and Fr-H2.

Genetic analyses have identified two loci in wheat and barley that mediate the capacity to overwinter in temperate climates. One locus co-segregates with VRN-1, which affects the vernalization requirement. This locus is known as Frost resistance-1 (Fr-1). The second locus, Fr-2, is coincident with a cluster of more than 12 Cbf genes. Cbf homologs in Arabidopsis thaliana play a key regulatory role in cold acclimatization and the acquisition of freezing tolerance. Here we report that the Hordeum vulgare (barley) locus VRN-H1/Fr-H1 affects expression of multiple barley Cbf genes at Fr-H2. RNA blot analyses, conducted on a 'Nure'x'Tremois' barley mapping population segregating for VRN-H1/Fr-H1 and Fr-H2, revealed that transcript levels of all cold-induced Cbf genes at Fr-H2 were significantly higher in recombinants harboring the vrn-H1 winter allele than in recombinants harboring the Vrn-H1 spring allele. Steady-state Cbf2 and Cbf4 levels were also significantly higher in recombinants harboring the Nure allele at Fr-H2. Additional experiments indicated that, in vrn-H1 genotypes requiring vernalization, Cbf expression levels were dampened after plants were vernalized, and dampened Cbf expression was accompanied by robust expression of Vrn-1. Cbf levels were also significantly higher in plants grown under short days than under long days. Experiments in wheat and rye indicated that similar regulatory mechanisms occurred in these plants. These results suggest that VRN-H1/Fr-H1 acts in part to repress or attenuate expression of the Cbf at Fr-H2; and that the greater level of low temperature tolerance attributable to the Nure Fr-H2 allele may be due to the greater accumulation of Cbf2 and Cbf4 transcripts during normal growth and development.

Validation of the VRN-H2/VRN-H1 epistatic model in barley reveals that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity.

The epistatic interaction of alleles at the VRN-H1 and VRN-H2 loci determines vernalization sensitivity in barley. To validate the current molecular model for the two-locus epistasis, we crossed homozygous vernalization-insensitive plants harboring a predicted "winter type" allele at either VRN-H1 (Dicktoo) or VRN-H2 (Oregon Wolfe Barley Dominant), or at both VRN-H (Calicuchima-sib) loci and measured the flowering time of unvernalized F(2) progeny under long-day photoperiod. We assessed whether the spring growth habit of Calicuchima-sib is an exception to the two-locus epistatic model or contains novel "spring" alleles at VRN-H1 (HvBM5A) and/or VRN-H2 (ZCCT-H) by determining allele sequence variants at these loci and their effects relative to growth habit. We found that (a) progeny with predicted "winter type" alleles at both VRN-H1 and VRN-H2 alleles exhibited an extremely delayed flowering (i.e. vernalization-sensitive) phenotype in two out of the three F(2) populations, (b) sequence flanking the vernalization critical region of HvBM5A intron 1 likely influences degree of vernalization sensitivity, (c) a winter habit is retained when ZCCT-Ha has been deleted, and (d) the ZCCT-H genes have higher levels of allelic polymorphism than other winterhardiness regulatory genes. Our results validate the model explaining the epistatic interaction of VRN-H2 and VRN-H1 under long-day conditions, demonstrate recovery of vernalization-sensitive progeny from crosses of vernalization-insensitive genotypes, show that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity, and provide molecular markers that are accurate predictors of "winter vs spring type" alleles at the VRN-H loci.

Mapping of barley homologs to genes that regulate low temperature tolerance in Arabidopsis.

We investigated the allelic nature and map locations of Hordeum vulgare (barley) homologs to three classes of Arabidopsis low temperature (LT) regulatory genes-CBFs, ICE1, and ZAT12-to determine if there were any candidates for winterhardiness-related quantitative trait loci (QTL). We phenotyped the Dicktoo x Morex (DxM) mapping population under controlled freezing conditions and in addition to the previously reported 5H-L Fr-H1 QTL, observed three additional LT tolerance QTLs on 1H-L, 4H-S, and 4H-L. We identified and assigned either linkage map or chromosome locations to 1 ICE1 homolog, 2 ZAT12 homologs, and 17 of 20 CBF homologs. Twelve of the CBF genes were located on 5H-L and the 11 with assigned linkage map positions formed 2 tandem clusters on 5H-L. A subset of these CBF genes was confirmed to be physically linked, validating the map position clustering. The tandem CBF clusters are not candidates for the DxM LT tolerance Fr-H1 QTL, as they are approximately 30 cM distal to the QTL peak. No LT tolerance QTL was detected in conjunction with the CBF gene clusters in Dicktoo x Morex. However, comparative mapping using common markers and BIN positions established the CBF clusters are coincident with reported Triticeae LT tolerance and COR gene accumulation QTLs and suggest one or more of the CBF genes may be candidates for Fr-H2 in some germplasm combinations. These results suggest members of the CBF gene family may function as components of winter-hardiness in the Triticeae and underscore both the importance of extending results from model systems to economically important crop species and in viewing QTL mapping results in the context of multiple germplasm combinations.

The CBF1-dependent low temperature signalling pathway, regulon and increase in freeze tolerance are conserved in Populus spp.

The meristematic tissues of temperate woody perennials must acclimate to freezing temperatures to survive the winter and resume growth the following year. To determine whether the C-repeat binding factor (CBF) family of transcription factors contributing to this process in annual herbaceous species also functions in woody perennials, we investigated the changes in phenotype and transcript profile of transgenic Populus constitutively expressing CBF1 from Arabidopsis (AtCBF1). Ectopic expression of AtCBF1 was sufficient to significantly increase the freezing tolerance of non-acclimated leaves and stems relative to wild-type plants. cDNA microarray experiments identified genes up-regulated by ectopic AtCBF1 expression in Populus, demonstrated a strong conservation of the CBF regulon between Populus and Arabidopsis and identified differences between leaf and stem regulons. We studied the induction kinetics and tissue specificity of four CBF paralogues identified from the Populus balsamifera subsp. trichocarpa genome sequence (PtCBFs). All four PtCBFs are cold-inducible in leaves, but only PtCBF1 and PtCBF3 show significant induction in stems. Our results suggest that the central role played by the CBF family of transcriptional activators in cold acclimation of Arabidopsis has been maintained in Populus. However, the differential expression of the PtCBFs and differing clusters of CBF-responsive genes in annual (leaf) and perennial (stem) tissues suggest that the perennial-driven evolution of winter dormancy may have given rise to specific roles for these 'master-switches' in the different annual and perennial tissues of woody species.

Molecular characterization of the duplicated meristem identity genes HvAP1a and HvAP1b in barley.

The vernalization gene VRN-1 has been identified as a MADS-box transcription factor orthologous to the meristem identity gene APETALA1 (AP1). A single copy of this gene was found in diploid wheat, but 2 copies were reported in barley. In this study, we present a detailed characterization of these 2 copies to understand their respective roles in the vernalization response. We identified 2 groups of barley bacterial artificial chromosomes (BACs), each containing 1 AP1 copy designated hereafter as HvAP1a and HvAP1b. A physical map of the VRN-H1 region showed that the HvAP1a BACs were part of the VRN-H1 region but that the HvAP1b BACs were not. Numerous structural changes were observed between the barley and wheat VRN-1 physical maps. In a population segregating for VRN-H1, the HvAP1a gene cosegregated with growth habit, suggesting that HvAP1a is the barley vernalization gene VRN-H1. The other copy, HvAP1b, was mapped on the centromeric region of chromosome 1H, the chromosome where vernalization gene VRN-H3 was previously mapped. We developed a mapping population segregating for VRN-H3 and showed that 2 molecular makers flanking HvAP1b locus were not linked to growth habit. The HvAP1b copy has a complete deletion of the first 2 exons, suggesting that it is a truncated pseudogene and not a candidate for VRN-H3. In summary, this study contributed a detailed physical map of the barley VRN-H1 region, showed several structural differences with the orthologous wheat region, and clarified the identity of the barley VRN-H1 gene.