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Tyrosinkinase-Inhibitoren (TKIs) haben die langfristigen Behandlungsergebnisse für Patienten mit chronischer myeloischer Leukämie (CML) erheblich verbessert. Nach Imatinib (einem TKI der ersten Generation) wurden TKIs der zweiten und dritten Generation entwickelt. Mit 5 TKIs, die auf BCR::ABL abzielen und in den meisten Ländern zugelassen sind (Imatinib, Dasatinib, Bosutinib, Nilotinib und Ponatinib), und der kürzlich erfolgten Zulassung von Asciminib in den USA sind die Behandlungsentscheidungen komplex und erfordern eine Bewertung patientenspezifischer Faktoren. Optimale Behandlungsstrategien für CML entwickeln sich weiter, wobei der Schwerpunkt verstärkt darauf liegt, ein tiefes molekulares Ansprechen zu erzielen. Anhand von klinisch relevanten Fallstudien, die von den Autoren dieses Reviews entwickelt wurden, diskutieren wir 3 Hauptszenarien aus der Perspektive internationaler Experten. Erstens untersucht dieser Übersichtsartikel patientenspezifische Merkmale, die die Entscheidungsfindung zwischen TKIs der ersten und zweiten Generation bei der Erstdiagnose von CML beeinflussen, einschließlich der Begleiterkrankungen des Patienten. Zweitens wird eine gründliche Bewertung der therapeutischen Optionen im Falle eines Versagens der Erstlinienbehandlung (wie in den Richtlinien des National Comprehensive Cancer Network und des European LeukemiaNet definiert) diskutiert, zusammen mit praktischen Erwägungen zur Überwachung des optimalen Ansprechens auf die TKI-Therapie. Drittens verdeutlicht diese Übersichtsarbeit die Erwägungen zum und die Bedeutung des Erreichen(s) einer behandlungsfreien Remission als Behandlungsziel. Aufgrund des Zeitpunkts der Erstellung befasst sich dieser Review auch mit den globalen Herausforderungen, denen Hämatologen bei der Behandlung von CML-Patienten während der COVID-19-Pandemie häufig gegenüberstehen. Da bei CML weiterhin neue Behandlungsansätze erforscht werden, erörtert diese Übersichtsarbeit schließlich auch das Aufkommen neuerer Therapien wie Asciminib. Dieser Artikel kann eine nützliche Referenz für Ärzte sein, die CML-Patienten mit TKI der zweiten Generation behandeln, und kann, da er sich auf die internationalen und persönlichen Erfahrungen der Ärzte konzentriert, einen Einblick in alternative, zuvor nicht in Betracht gezogene Ansätze geben.

Ponatinib, the only third-generation pan-BCR::ABL1 inhibitor with activity against all known BCR::ABL1 mutations including T315I, has demonstrated deep and durable responses in patients with chronic-phase chronic myeloid leukemia (CP-CML) resistant to prior second-generation (2G) TKI treatment. We present efficacy and safety outcomes from the Ponatinib Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and CML Evaluation (PACE) and Optimizing Ponatinib Treatment in CP-CML (OPTIC) trials for this patient population. PACE (NCT01207440) evaluated ponatinib 45 mg/day in CML patients with resistance to prior TKI or T315I. In OPTIC (NCT02467270), patients with CP-CML and resistance to ≥2 prior TKIs or T315I receiving 45 or 30 mg/day reduced their doses to 15 mg/day upon achieving ≤1% BCR::ABL1

Introduction: Patients (pts) with primary and secondary myelofibrosis (MF) have a reduced life expectancy. While several oral Janus Kinase (JAK)-1/2 inhibitors such as ruxolitinib (RUX) have demonstrated improvements in symptom burden and spleen volume reduction compared to physician choice of best available therapy leading to their approval by the US FDA, responses to these agents are generally time-limited. Additionally, these agents are not considered to be disease-modifying and survival in pts after RUX discontinuation is poor with a median survival of 11-14 months. Thus, novel mechanism-based therapies for pts with suboptimal response or progression on RUX are needed. Preclinical Rationale: JAK2 V617F mutations promote transition from G1 to S-phase of the cell cycle via increased expression of CDC25A in preclinical models and CDC25A expression is upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D also play a role in MF pathogenesis and drive myeloproliferation. Finally, combination of the CDK4/6 inhibitor ribociclib and RUX demonstrated reduction in spleen and liver size when compared to RUX alone in murine models, suggesting synergy (Rampal et al., CCR 2021). Methods: This multicenter, phase I dose-escalation trial evaluates the safety of RUX + the CDK4/6 inhibitor abemaciclib in pts with primary or secondary MF with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to RUX. An inadequate response will be defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms [presence of 1 symptom score ≥5 or 2 symptom scores ≥3 using the screening MPN-SAF TSS. This study follows a conventional “3+3” dose-escalation design, in which pts on a stable dose of RUX (10mg or 15mg BID) with sufficient baseline platelet and absolute neutrophil count will be treated with increasing doses of abemaciclib. Abemaciclib was chosen due to less myelosuppression compared with other CDK4/6 inhibitors. The pre-study dose of ruxolitinib will be maintained to determine the maximum tolerated dose of the combination of RUX+ abemaciclib. The primary endpoint is to determine the safety and tolerability, and to identify the recommended phase II dose of abemaciclib in MF pts on a stable dose of RUX. Secondary endpoints include overall response rate, progression-free and overall survival, and duration of response per the modified International Working Group - Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and the European Leukemia Net (ELN) 2013 consensus definitions. Correlative studies are planned to identify biomarkers predictive of response to RUX + abemaciclib, mechanisms of resistance (e.g., MAP kinase pathway activation), and any disease-modifying effects of combination therapy. Citation Format: Jan P. Bewersdorf, Srdan Verstovsek, Andriy Derkach, Lucia Masarova, Naveen Pemmaraju, Eytan M. Stein, Michael Mauro, Raajit Rampal, Prithviraj Bose. A phase I study of ruxolitinib in combination with abemaciclib for patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT242.

SummaryNuclear pore complexes (NPCs) are large protein assemblies that facilitate transport of macromolecules across the nuclear envelope (NE) [1, 2]. How thousands of NPCs rapidly assemble after open mitosis to form a functional NE is not known. Recruitment of the Nup107-160 outer ring scaffold to chromatin initiates NPC assembly. The Nup53/93 complex bridges the outer ring to the central channel to form a functional pore [3–6]. Nup53 interacts with the conserved transmembrane nucleoporin Ndc1; however, how Ndc1 contributes to post-mitotic NPC assembly is unclear [7–9]. Here, we use C. elegans embryos to show that the timely formation of a functional NE after mitosis depends on Ndc1. Endogenously tagged Ndc1 is recruited early to the reforming NE and is highly mobile in the nuclear rim. 3D analysis of post-mitotic NE formation revealed a decreased NPC density in NEs of ndc1 deleted embryos – continuous nuclear membranes contained few holes where assembling NPCs are normally located. Nup160 is highly mobile in NEs depleted of Ndc1 and outer ring scaffold components are less enriched at the rim. When both ndc1 and nup53 are absent, nuclear assembly fails. Together, these data show that Ndc1 dynamically associates with the NE and promotes stable association of the outer ring scaffold in the NE to facilitate NPC assembly after open mitosis. Furthermore, Ndc1 and Nup53 function in parallel to drive nuclear assembly. We propose that Ndc1 is a dynamic membrane adaptor that helps recruit and promote the self-assembly of the nuclear pore scaffold to drive post-mitotic NPC assembly.

Objective: The purpose of this study was to investigate the morphological changes of round window membrane (RWM) in chinchillas with Streptococcus pneumoniae (S. pneumoniae) serotype 7F induced acute otitis media (AOM) by two dimensional (2D) and three dimensional (3D) measurements. Methods: Temporal bone specimens taken from 12 chinchillas were divided into two groups. The control group consisted of healthy animals that were injected with intrabullar saline. The subjects in the experimental group were induced with AOM by intrabullar injection of S. pneumoniae 7F. The 2D and 3D measurements of RWM were compared between the groups. Results: Dramatic changes were noted in the RWM of the experimental group compared to the control group. The thickness [mean ± standard deviation (SD)] of the RWM was significantly (p

The nuclear permeability barrier depends on closure of nuclear envelope holes. Penfield et al. show that the CNEP-1–lipin pathway limits production of ER membranes that feed into and close nuclear envelope holes. Upon excess membrane production, remodeling by nuclear envelope adaptors to ESCRT-III contributes to nuclear closure., The nuclear permeability barrier depends on closure of nuclear envelope (NE) holes. Here, we investigate closure of the NE opening surrounding the meiotic spindle in C. elegans oocytes. ESCRT-III components accumulate at the opening but are not required for nuclear closure on their own. 3D analysis revealed cytoplasmic membranes directly adjacent to NE holes containing meiotic spindle microtubules. We demonstrate that the NE protein phosphatase, CNEP-1/CTDNEP1, controls de novo glycerolipid synthesis through lipin to prevent invasion of excess ER membranes into NE holes and a defective NE permeability barrier. Loss of NE adaptors for ESCRT-III exacerbates ER invasion and nuclear permeability defects in cnep-1 mutants, suggesting that ESCRTs restrict excess ER membranes during NE closure. Restoring glycerolipid synthesis in embryos deleted for CNEP-1 and ESCRT components rescued NE permeability defects. Thus, regulating the production and feeding of ER membranes into NE holes together with ESCRT-mediated remodeling is required for nuclear closure.

The nuclear permeability barrier depends on closure of holes in the nuclear envelope (NE). Here, we use meiotic C. elegans oocytes to demonstrate that local control of glycerophospholipid synthesis by CNEP-1/CTDNEP1 regulates the insertion of ER sheets into NE holes and functions independently of ESCRT-III to ensure NE closure. Deletion of CNEP-1 causes excess incorporation of ER membranes into NE holes and a defective NE permeability barrier. ESCRT-III components accumulate at the NE opening surrounding the meiotic spindle, and loss of NE adaptors for ESCRT-III exacerbates NE sealing defects in cnep-1 mutants. Limiting ER sheet production by restoring glycerophospholipid synthesis in cnep-1 mutants rescued NE permeability defects. 3D analysis showed that membrane sheets feed into and narrow NE holes occluded by meiotic spindle microtubules supporting a role for ER sheet insertion in NE closure. Thus, feeding of ER sheets into NE holes must be coordinated with production of ER sheets near the NE to promote NE closure.

We developed a first-of-kind dasatinib-derivative imaging agent, (18)F-SKI-249380 ((1)(8)F-SKI), and validated its use for noninvasive in vivo tyrosine kinase-targeted tumor detection in preclinical models. In this study, we assessed the feasibility of using (18)F-SKI for PET imaging in patients with malignancies. Methods: Five patients with a prior diagnosis of breast cancer, renal cell cancer, or leukemia underwent whole-body PET/CT imaging 90 min after injection of (18)F-SKI (mean, 241.24 ± 116.36 MBq) as part of a prospective study. In addition, patients underwent either a 30-min dynamic scan of the upper abdomen including, at least partly, cardiac left ventricle, liver, spleen, and kidney (n = 2) or three 10-min whole-body PET/CT scans (n = 3) immediately after injection and blood-based radioactivity measurements to determine the time course of tracer distribution and facilitate radiation dose estimates. A subset of 3 patients had a delayed whole-body PET/CT scan at 180 min. Biodistribution, dosimetry, and tumor uptake were quantified. Absorbed doses were calculated using OLINDA/EXM 1.0. Results: No adverse events occurred after injection of (18)F-SKI. In total, 27 tumor lesions were analyzed, with a median SUV(peak) of 1.4 (range, 0.7–2.3) and tumor-to-blood ratios of 1.6 (range, 0.8–2.5) at 90 min after injection. The intratumoral drug concentrations calculated for 4 reference lesions ranged from 0.03 to 0.07 nM. In all reference lesions, constant tracer accumulation was observed between 30 and 90 min after injection. A blood radioassay indicated that radiotracer clearance from blood and plasma was initially rapid (blood half-time, 1.31 ± 0.81 min; plasma, 1.07 ± 0.66 min; n = 4), followed variably by either a prolonged terminal phase (blood half-time, 285 ± 148.49 min; plasma, 240 ± 84.85 min; n = 2) or a small rise to a plateau (n = 2). Like dasatinib, (18)F-SKI underwent extensive metabolism after administration, as evidenced by metabolite analysis. Radioactivity was predominantly cleared via the hepatobiliary route. The highest absorbed dose estimates (mGy/MBq) in normal tissues were to the right colon (0.167 ± 0.04) and small intestine (0.153 ± 0.03). The effective dose was 0.0258 mSv/MBq (SD, 0.0034 mSv/MBq). Conclusion: (18)F-SKI demonstrated significant tumor uptake, distinct image contrast despite low injected doses, and rapid clearance from blood.

Transient nuclear envelope (NE) ruptures in the Caenorhabditis elegans zygote are caused by a weakened nuclear lamina during nuclear positioning. Dynein-pulling forces enhance the severity of ruptures, while lamin restricts nucleocytoplasmic mixing and allows stable NE repair. This work is the first mechanistic analysis of NE rupture and repair in an organism., Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) ruptures and repairs in interphase. The duration of NE ruptures depends on lamins; however, the underlying mechanisms and relevance to in vivo events are not known. Here, we use the Caenorhabditis elegans zygote to analyze lamin’s role in NE rupture and repair in vivo. Transient NE ruptures and subsequent NE collapse are induced by weaknesses in the nuclear lamina caused by expression of an engineered hypomorphic C. elegans lamin allele. Dynein-generated forces that position nuclei enhance the severity of transient NE ruptures and cause NE collapse. Reduction of dynein forces allows the weakened lamin network to restrict nucleo–cytoplasmic mixing and support stable NE recovery. Surprisingly, the high incidence of transient NE ruptures does not contribute to embryonic lethality, which is instead correlated with stochastic chromosome scattering resulting from premature NE collapse, suggesting that C. elegans tolerates transient losses of NE compartmentalization during early embryogenesis. In sum, we demonstrate that lamin counteracts dynein forces to promote stable NE repair and prevent catastrophic NE collapse, and thus provide the first mechanistic analysis of NE rupture and repair in an organismal context.

Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) undergoes ruptures leading to transient mixing of nuclear and cytoplasmic components. The duration of transient NE ruptures depends on lamins, however the underlying mechanisms and the relevance toin vivoevents is not known. Here, we useC. elegansembryos to show that dynein forces that position nuclei increase the severity of lamin-induced NE rupturesin vivo. In the absence of dynein forces, lamin prevents nuclear-cytoplasmic mixing caused by NE ruptures. By monitoring the dynamics of NE rupture events, we demonstrate that lamin is required for a distinct phase in NE recovery that restricts nucleocytoplasmic mixing prior to the full restoration of NE rupture sites. We show that laser-induced puncture of the NE recapitulates phenotypes associated with NE recovery in wild type cells. Surprisingly, we find that embryonic lethality does not correlate with the incidence of NE rupture events suggesting that embryos survive transient losses of NE compartmentalization during early embryogenesis. In addition to presenting the first mechanistic analysis of transient NE rupturesin vivo, this work demonstrates that lamin controls the duration of NE ruptures by opposing dynein forces on ruptured nuclei to allow reestablishment of the NE permeability barrier and subsequent restoration of NE rupture sites.

mel-28 (maternal-effect-lethal-28) encodes a conserved protein required for nuclear envelope function and chromosome segregation in Caenorhabditis elegans. Because mel-28 is a strict maternal-effect lethal gene, its function is required in the early embryo but appears to be dispensable for larval development. We wanted to test the idea that mel-28 has postembryonic roles that are buffered by the contributions of other genes. To find genes that act coordinately with mel-28, we did an RNA interference−based genetic interaction screen using mel-28 and wild-type larvae. We screened 18,364 clones and identified 65 genes that cause sterility in mel-28 but not wild-type worms. Some of these genes encode components of the nuclear pore. In addition we identified genes involved in dynein and dynactin function, vesicle transport, and cell-matrix attachments. By screening mel-28 larvae we have bypassed the requirement for mel-28 in the embryo, uncovering pleiotropic functions for mel-28 later in development that are normally provided by other genes. This work contributes toward revealing the gene networks that underlie cellular processes and reveals roles for a maternal-effect lethal gene later in development.

Resumen del póster presentado al 11th Meeting of the Spanish Society for Developmental Biology, celebrado en Girona (España) del 19 al 21 de octubre de 2016.-- et al., Nucleoporins are the constituents of nuclear pore complexes (NPCs) and are essential regulators of nucleocytoplasmic transport, gene expression and genome stability. The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex and is required for correct segregation of mitotic chromosomes. MEL-28 has a dynamic behavior: it localizes to nuclear pore complexes and chromatin during interphase and shuttles to kinetochores during cell division. However, it is unknown how MEL-28 localization and activity is regulated. Here we present a systematic functional and structural analysis of MEL-28 in C. elegans early development and human ELYS in cultured cells. We have identified functional domains responsible for NPC and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation. Surprisingly, we found that perturbations to MEL-28’s conserved AT-hook domain do not affect MEL-28 localization although they disrupt MEL-28 function and delay cell cycle progression in a DNA damage checkpoint-dependent manner. Our analyses also uncover a novel meiotic role of MEL-28. Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation. To understand the function of MEL-28 chromatin binding we have used DamID to identify the chromatin regions with which MEL-28 associates. Interestingly, MEL-28 is enriched at transcribed genes and correlates positively with active histone marks, suggesting that it may be involved in regulation of gene expression. We compared the MEL-28 chromatin profile with the profile of another nucleoporin, NPP-22, which is permanently anchored to the nuclear pore complex. Surprisingly, we found that the chromatin association profile of NPP-22 was more similar to the profile of the nuclear lamina protein LMN-1 than to MEL-28’s profile, suggesting that individual NPPs interact with specific chromatin domains. Finally, GO-term analysis reveals that MEL-28-associated genes are related to larval and reproductive development. This suggests that MEL-28 has postembryonic functions that have not yet been studied.