Immunoproteasome function maintains oncogenic gene expression in KMT2A-complex driven leukemia.

Pharmacologic targeting of chromatin-associated protein complexes has shown significant responses in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) but resistance frequently develops to single agents. This points to a need for therapeutic combinations that target multiple mechanisms. To enhance our understanding of functional dependencies in KMT2A-r AML, we have used a proteomic approach to identify the catalytic immunoproteasome subunit PSMB8 as a specific vulnerability. Genetic and pharmacologic inactivation of PSMB8 results in impaired proliferation of murine and human leukemic cells while normal hematopoietic cells remain unaffected. Disruption of immunoproteasome function drives an increase in transcription factor BASP1 which in turn represses KMT2A-fusion protein target genes. Pharmacologic targeting of PSMB8 improves efficacy of Menin-inhibitors, synergistically reduces leukemia in human xenografts and shows preserved activity against Menin-inhibitor resistance mutations. This identifies and validates a cell-intrinsic mechanism whereby selective disruption of proteostasis results in altered transcription factor abundance and repression of oncogene-specific transcriptional networks. These data demonstrate that the immunoproteasome is a relevant therapeutic target in AML and that targeting the immunoproteasome in combination with Menin-inhibition could be a novel approach for treatment of KMT2A-r AML.

Proteasome inhibition potentiates Kv1.3 potassium channel expression as therapeutic target in drug-sensitive and -resistant human melanoma cells.

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.

T cell-specific constitutive active SHP2 enhances T cell memory formation and reduces T cell activation.

Upon antigen recognition by the T cell receptor (TCR), a complex signaling network orchestrated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) regulates the transmission of the extracellular signal to the nucleus. The role of the PTPs Src-homology 2 (SH2) domain-containing phosphatase 1 (SHP1, Ptpn6) and Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2, Ptpn11) have been studied in various cell types including T cells. Whereas SHP1 acts as an essential negative regulator of the proximal steps in T cell signalling, the role of SHP2 in T cell activation is still a matter of debate. Here, we analyzed the role of the constitutively active SHP2-D61Y-mutant in T cell activation using knock-in mice expressing the mutant form Ptpn11D61Y in T cells. We observed reduced numbers of CD8+ and increased numbers of CD4+ T cells in the bone marrow and spleen of young and aged SHP2-D61Y-mutant mice as well as in Influenza A Virus (IAV)-infected mice compared to controls. In addition, we found elevated frequencies of effector memory CD8+ T cells and an upregulation of the programmed cell death protein 1 (PD-1)-receptor on both CD4+ and CD8+ T cells. Functional analysis of SHP2-D61Y-mutated T cells revealed an induction of late apoptosis/necrosis, a reduced proliferation and altered signaling upon TCR stimulation. However, the ability of D61Y-mutant mice to clear viral infection was not affected. In conclusion, our data indicate an important regulatory role of SHP2 in T cell function, where the effect is determined by the kinetics of SHP2 phosphatase activity and differs in the presence of the permanently active and the temporally regulated phosphatase. Due to interaction of SHP2 with the PD-1-receptor targeting the protein-tyrosine phosphatase might be a valuable tool to enhance T cell activities in immunotherapy.

Recycling and Reshaping-E3 Ligases and DUBs in the Initiation of T Cell Receptor-Mediated Signaling and Response.

T cell activation plays a central role in supporting and shaping the immune response. The induction of a functional adaptive immune response requires the control of signaling processes downstream of the T cell receptor (TCR). In this regard, protein phosphorylation and dephosphorylation have been extensively studied. In the past decades, further checkpoints of activation have been identified. These are E3 ligases catalyzing the transfer of ubiquitin or ubiquitin-like proteins to protein substrates, as well as specific peptidases to counteract this reaction, such as deubiquitinating enzymes (DUBs). These posttranslational modifications can critically influence protein interactions by targeting proteins for degradation by proteasomes or mediating the complex formation required for active TCR signaling. Thus, the basic aspects of T cell development and differentiation are controlled by defining, e.g., the threshold of activation in positive and negative selection in the thymus. Furthermore, an emerging role of ubiquitination in peripheral T cell tolerance has been described. Changes in the function and abundance of certain E3 ligases or DUBs involved in T cell homeostasis are associated with the development of autoimmune diseases. This review summarizes the current knowledge of E3 enzymes and their target proteins regulating T cell signaling processes and discusses new approaches for therapeutic intervention.

Streptococcus pneumoniae and Influenza A Virus Co-Infection Induces Altered Polyubiquitination in A549 Cells.

Epithelial cells are an important line of defense within the lung. Disruption of the epithelial barrier by pathogens enables the systemic dissemination of bacteria or viruses within the host leading to severe diseases with fatal outcomes. Thus, the lung epithelium can be damaged by seasonal and pandemic influenza A viruses. Influenza A virus infection induced dysregulation of the immune system is beneficial for the dissemination of bacteria to the lower respiratory tract, causing bacterial and viral co-infection. Host cells regulate protein homeostasis and the response to different perturbances, for instance provoked by infections, by post translational modification of proteins. Aside from protein phosphorylation, ubiquitination of proteins is an essential regulatory tool in virtually every cellular process such as protein homeostasis, host immune response, cell morphology, and in clearing of cytosolic pathogens. Here, we analyzed the proteome and ubiquitinome of A549 alveolar lung epithelial cells in response to infection by either Streptococcus pneumoniae D39Δcps or influenza A virus H1N1 as well as bacterial and viral co-infection. Pneumococcal infection induced alterations in the ubiquitination of proteins involved in the organization of the actin cytoskeleton and Rho GTPases, but had minor effects on the abundance of host proteins. H1N1 infection results in an anti-viral state of A549 cells. Finally, co-infection resembled the imprints of both infecting pathogens with a minor increase in the observed alterations in protein and ubiquitination abundance.

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines.

Vector-based SARS-CoV-2 vaccines have been associated with vaccine- induced thrombosis with thrombocytopenia syndrome (VITT/TTS), but the causative factors are still unresolved. We comprehensively analyzed the ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Johnson and Johnson) vaccines. ChAdOx1 nCoV-19 contains significant amounts of host cell protein impurities, including functionally active proteasomes, and adenoviral proteins. A much smaller amount of impurities was found in Ad26.COV2.S. Platelet factor 4 formed complexes with ChAdOx1 nCoV-19 constituents, but not with purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not by Ad26.COV2.S. These differences in impurities together with EDTAinduced capillary leakage might contribute to the higher incidence rate of VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.

Staphylococcus aureus nasal colonization among dental health care workers in Northern Germany (StaphDent study).

Methicillin-resistant Staphylococcus aureus (MRSA) can colonize dental patients and students, however, studies on the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) among dental health care workers (DHCW) including use of personal protective equipment (PPE) are scarce. We conducted an observational study (StaphDent study) to (I) determine the prevalence of MRSA and MSSA colonization in DHCW in the region of Mecklenburg Western-Pomerania, Germany, (II) resolve the S. aureus population structure to gain hints on possible transmission events between co-workers, and (III) clarify use of PPE. Nasal swabs were obtained from dentists (n = 149), dental assistants (n = 297) and other dental practice staff (n = 38). Clonal relatedness of MSSA isolates was investigated using spa typing and, in some cases, whole genome sequencing (WGS). PPE use was assessed by questionnaire. While 22.3% (108/485) of the participants were colonized with MSSA, MRSA was not detected. MSSA prevalence was not associated with size of dental practices, gender, age, or duration of employment. The identified 61 spa types grouped into 17 clonal complexes and four sequence types. Most spa types (n = 47) were identified only once. In ten dental practices one spa type occurred twice. WGS data analysis confirmed a close clonal relationship for 4/10 isolate pairs. PPE was regularly used by most dentists and assistants. To conclude, the failure to recover MRSA from DHCW reflects the low MRSA prevalence in this region. Widespread PPE use suggests adherence to routine hygiene protocols. Compared to other regional HCW MRSA rates the consequent usage of PPE seems to be protective.

The platelet proteasome and immunoproteasome are stable in buffy-coat derived platelet concentrates for up to 7 days.

OBJECTIVES: Characterization of the proteasome and its stability in buffy-coat derived platelet concentrates (PCs) during storage. BACKGROUND: The proteasome plays a key role in cell homeostasis by processing misfolded or abnormal proteins and regulating the levels and activities of a high number of proteins contributing to cell cycle, survival, and proliferation. Controversial data exist, whether inhibition of the proteasome affects platelet function. Little is known about function, expression, and stability of the proteasome in PCs during storage, and the potential role of the platelet proteasome in storage lesions. STUDY DESIGN AND METHODS: PCs were produced by the buffy-coat method in additive solution and stored at room temperature under agitation. Platelet aggregation was monitored by light transmission aggregometry. Proteasome complexes were assessed by immunoprecipitation and immunoblotting, and proteasome activity was measured using fluorogenic substrates specific for the three different proteolytic activities over 7 days of storage. RESULTS: Proteasome inhibition led to a decreased platelet aggregation response after activation with collagen, ADP, TRAP-6, and thrombin. There were no changes in the expression of the catalytic active subunits as well as the proteasome activity during storage of PCs, comparing baseline and day 7. DISCUSSION: Platelet proteasome function is relevant for platelet aggregation in response to various agonists. The constitutive and stable expression of the active standard- and immunoproteasome in platelets makes it unlikely that loss of proteasome function is a relevant cause of storage lesions.

The Immunoproteasome Subunits LMP2, LMP7 and MECL-1 Are Crucial Along the Induction of Cerebral Toxoplasmosis.

Cell survival and function critically relies on the fine-tuned balance of protein synthesis and degradation. In the steady state, the standard proteasome is sufficient to maintain this proteostasis. However, upon inflammation, the sharp increase in protein production requires additional mechanisms to limit protein-associated cellular stress. Under inflammatory conditions and the release of interferons, the immunoproteasome (IP) is induced to support protein processing and recycling. In antigen-presenting cells constitutively expressing IPs, inflammation-related mechanisms contribute to the formation of MHC class I/II-peptide complexes, which are required for the induction of T cell responses. The control of Toxoplasma gondii infection relies on Interferon-γ (IFNγ)-related T cell responses. Whether and how the IP affects the course of anti-parasitic T cell responses along the infection as well as inflammation of the central nervous system is still unknown. To answer this question we used triple knockout (TKO) mice lacking the 3 catalytic subunits of the immunoproteasome (ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7). Here we show that the numbers of dendritic cells, monocytes and CD8+ T cells were reduced in Toxoplasma gondii-infected TKO mice. Furthermore, impaired IFNγ, TNF and iNOS production was accompanied by dysregulated chemokine expression and altered immune cell recruitment to the brain. T cell differentiation was altered, apoptosis rates of microglia and monocytes were elevated and STAT3 downstream signaling was diminished. Consequently, anti-parasitic immune responses were impaired in TKO mice leading to elevated T. gondii burden and prolonged neuroinflammation. In summary we provide evidence for a critical role of the IP subunits ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 for the control of cerebral Toxoplasma gondii infection and subsequent neuroinflammation.

Targeting the innate immunoreceptor RIG-I overcomes melanoma-intrinsic resistance to T cell immunotherapy.

Understanding tumor resistance to T cell immunotherapies is critical to improve patient outcomes. Our study revealed a role for transcriptional suppression of the tumor-intrinsic HLA class I (HLA-I) antigen processing and presentation machinery (APM) in therapy resistance. Low HLA-I APM mRNA levels in melanoma metastases before immune checkpoint blockade (ICB) correlated with nonresponsiveness to therapy and poor clinical outcome. Patient-derived melanoma cells with silenced HLA-I APM escaped recognition by autologous CD8+ T cells. However, targeted activation of the innate immunoreceptor RIG-I initiated de novo HLA-I APM transcription, thereby overcoming T cell resistance. Antigen presentation was restored in interferon-sensitive (IFN-sensitive) but also immunoedited IFN-resistant melanoma models through RIG-I-dependent stimulation of an IFN-independent salvage pathway involving IRF1 and IRF3. Likewise, enhanced HLA-I APM expression was detected in RIG-Ihi (DDX58hi) melanoma biopsies, correlating with improved patient survival. Induction of HLA-I APM by RIG-I synergized with antibodies blocking PD-1 and TIGIT inhibitory checkpoints in boosting the antitumor T cell activity of ICB nonresponders. Overall, the herein-identified IFN-independent effect of RIG-I on tumor antigen presentation and T cell recognition proposes innate immunoreceptor targeting as a strategy to overcome intrinsic T cell resistance of IFN-sensitive and IFN-resistant melanomas and improve clinical outcomes in immunotherapy.

Structure and function of the ubiquitin-proteasome system in platelets.

Platelets are small anucleate blood cells with a life span of 7 to 10 days. They are main regulators of hemostasis. Balanced platelet activity is crucial to prevent bleeding or occlusive thrombus formation. Growing evidence supports that platelets also participate in immune reactions, and interaction between platelets and leukocytes contributes to both thrombosis and inflammation. The ubiquitin-proteasome system (UPS) plays a key role in maintaining cellular protein homeostasis by its ability to degrade non-functional self-, foreign, or short-lived regulatory proteins. Platelets express standard and immunoproteasomes. Inhibition of the proteasome impairs platelet production and platelet function. Platelets also express major histocompatibility complex (MHC) class I molecules. Peptide fragments released by proteasomes can bind to MHC class I, which makes it also likely that platelets can activate epitope specific cytotoxic T lymphocytes (CTLs). In this review, we focus on current knowledge on the significance of the proteasome for the functions of platelets as critical regulators of hemostasis as well as modulators of the immune response.

Influence of Bio-sorb® Cream on Sweat Production and Efficacy of Surgical Hand Antisepsis Under Surgical Gloves.

Aim: Since the introduction of surgical gloves, one of the main challenges has been to improve donning and wearability. For the wearer, the formation of "glove juice" is problematic. To improve gliding properties for donning the gloves and absorbing sweat, in 1963 Bio-sorb® cream: sterile powder cream (Johnson & Johnson Medical, Gargrave, Skipton, United Kingdom) was introduced. Individuals subjectively reported a reduction of sweat production inside the glove, however, the actual effect on sweat accumulation and surgical hand antisepsis has not been examined. Methods: Twenty-six volunteers were used in a study designed to examine the effect of a sterile hand cream applied after surgical hand antisepsis on sweat accumulation inside surgical gloves. A woven cotton glove was worn underneath the surgical glove. Weight differences were used to determine the amount of sweat produced. The influence of Bio-sorb on the efficacy of surgical hand antisepsis was tested immediately and after 90 minutes of wear time by the sampling technique as per EN 12791 and by bacterial analysis of glove juice according to tentative final monograph 1994. Results and Discussion: The amount of sweat produced inside the glove was not reduced by the cream (1.07 ± 0.5 g versus control 1.03 ± 0.5 g; p = 0.75). Considering different skin conditions, it may be possible that some wearers might subjectively observe decreased sweat production after using Bio-sorb cream before donning surgical gloves, because sweat production did decrease in 10 of 26 test subjects. Bio-sorb cream did not affect the efficacy of surgical hand antisepsis either immediately or after 90 minutes. Conclusion: Because of possible risks of contamination of the surgical site with cornstarch from Bio-sorb cream in the case of a glove breach and the failed statistical proof of reduced sweat production, the use of Bio-sorb cream should not to be recommended.

ER-aminopeptidase 1 determines the processing and presentation of an immunotherapy-relevant melanoma epitope.

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100209-217 epitope, which is liberated from the melanoma differentiation antigen gp100PMEL17 , is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)-but not ERAP2-defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100209-217 -containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.

Prevention of Cervical Cancer: Guideline of the DGGG and the DKG (S3 Level, AWMF Register Number 015/027OL, December 2017) - Part 1 with Introduction, Screening and the Pathology of Cervical Dysplasia.

Aims Annual opportunistic screening for cervical carcinoma has been carried out in Germany since 1971. The creation of this S3 guideline meets an important need, outlined in the National Cancer Plan, with regard to screening for cervical cancer, as the guideline aims to provide important information and support for planned organized screening for cervical cancer in Germany. Methods With the financial support of German Cancer Aid, 21 professional societies developed evidence-based statements and recommendations (classified using the GRADE system) for the screening, management and treatment of precancerous conditions of the cervix. Two independent scientific institutes compiled systematic reviews for this guideline. Recommendations The first part of this short summary presents the pathological basis and considers various questions related to screening for cervical cancer. As also reported in earlier reviews, the meta-analysis by Kleijnen Systematic Reviews showed that HPV-based screening offers better protection against invasive cervical cancer compared to cytology-based screening. The authors of this guideline therefore recommend - in accordance with the guideline of the Joint National Committee of Germany (Gemeinsamer Bundesauschuss, G-BA) - that women aged 35 and above should be examined at regular intervals (at least every 3 years) and undergo HPV-based screening. Co-testing can also be carried out. Women between the ages of 20 and 35 should have cytological screening every 2 years.

Prevention of Cervical Cancer: Guideline of the DGGG and the DKG (S3 Level, AWMF Register Number 015/027OL, December 2017) - Part 2 on Triage, Treatment and Follow-up.

Aims Annual opportunistic screening for cervical carcinoma has been done in Germany since 1971. The creation of this S3 guideline meets an important need, outlined in the National Cancer Plan, with regard to screening for cervical cancer, as this guideline aims to provide important information and support for planned organized screening for cervical cancer in Germany. Methods With the financial support of German Cancer Aid, 21 professional societies developed evidence-based statements and recommendations (classified using the GRADE system) for the screening, management and treatment of precancerous conditions of the cervix. Two independent scientific institutes compiled systematic reviews for this guideline. Recommendations The second part of this short summary deals with the triage, treatment and follow-up care of cervical dysplasia. With regard to those women who do not participate in screening, the guideline authors recommend sending out repeat invitation letters or an HPV self-collection kit. Colposcopy should be carried out for further investigation if cytology findings are Pap II-p and HPV test results are positive or if the results of an HPV 16 or HPV 18 screening test are positive. A single abnormal Pap smear should be triaged and investigated using HPV testing or p16/Ki67 dual staining.

Deficiency of the immunoproteasome subunit ß5i/LMP7 supports the anxiogenic effects of mild stress and facilitates cued fear memory in mice.

Proteolysis as mediated by one of the major cellular protein degradation pathways, the ubiquitin-proteasome system (UPS), plays an essential role in learning and memory formation. However, the functional relevance of immunoproteasomes in the healthy brain and especially their impact on normal brain function including processes of learning and memory has not been investigated so far. In the present study, we analyzed the phenotypic effects of an impaired immunoproteasome formation using a ß5i/LMP7-deficient mouse model in different behavioral paradigms focusing on locomotor activity, exploratory behavior, innate anxiety, startle response, prepulse inhibition, as well as fear and safety conditioning. Overall, our results demonstrate no strong effects of constitutive ß5i/LMP7-deficiency on gross locomotor abilities and anxiety-related behavior in general. However, ß5i/LMP7-deficient mice expressed more anxiety after mild stress and increased cued fear after fear conditioning. These findings indicate that the basal proper formation of immunoproteasomes and/or at least the expression of ß5i/LMP7 in healthy mice seem to be involved in the regulation of anxiety and cued fear levels.

Memantine potentiates cytarabine-induced cell death of acute leukemia correlating with inhibition of Kv1.3 potassium channels, AKT and ERK1/2 signaling.

BACKGROUND: Treatment of acute leukemia is challenging and long-lasting remissions are difficult to induce. Innovative therapy approaches aim to complement standard chemotherapy to improve drug efficacy and decrease toxicity. Promising new therapeutic targets in cancer therapy include voltage-gated Kv1.3 potassium channels, but their role in acute leukemia is unclear. We reported that Kv1.3 channels of lymphocytes are blocked by memantine, which is known as an antagonist of neuronal N-methyl-D-aspartate type glutamate receptors and clinically applied in therapy of advanced Alzheimer disease. Here we evaluated whether pharmacological targeting of Kv1.3 channels by memantine promotes cell death of acute leukemia cells induced by chemotherapeutic cytarabine. METHODS: We analyzed acute lymphoid (Jurkat, CEM) and myeloid (HL-60, Molm-13, OCI-AML-3) leukemia cell lines and patients' acute leukemic blasts after treatment with either drug alone or the combination of cytarabine and memantine. Patch-clamp analysis was performed to evaluate inhibition of Kv1.3 channels and membrane depolarization by memantine. Cell death was determined with propidium iodide, Annexin V and SYTOX staining and cytochrome C release assay. Molecular effects of memantine co-treatment on activation of Caspases, AKT, ERK1/2, and JNK signaling were analysed by Western blot. Kv1.3 channel expression in Jurkat cells was downregulated by shRNA. RESULTS: Our study demonstrates that memantine inhibits Kv1.3 channels of acute leukemia cells and in combination with cytarabine potentiates cell death of acute lymphoid and myeloid leukemia cell lines as well as primary leukemic blasts from acute leukemia patients. At molecular level, memantine co-application fosters concurrent inhibition of AKT, S6 and ERK1/2 and reinforces nuclear down-regulation of MYC, a common target of AKT and ERK1/2 signaling. In addition, it augments mitochondrial dysfunction resulting in enhanced cytochrome C release and activation of Caspase-9 and Caspase-3 leading to amplified apoptosis. CONCLUSIONS: Our study underlines inhibition of Kv1.3 channels as a therapeutic strategy in acute leukemia and proposes co-treatment with memantine, a licensed and safe drug, as a potential approach to promote cytarabine-based cell death of various subtypes of acute leukemia.

Evolution of melanoma cross-resistance to CD8+ T cells and MAPK inhibition in the course of BRAFi treatment.

The profound but frequently transient clinical responses to BRAFV600 inhibitor (BRAFi) treatment in melanoma emphasize the need for combinatorial therapies. Multiple clinical trials combining BRAFi and immunotherapy are under way to further enhance therapeutic responses. However, to which extent BRAFV600 inhibition may affect melanoma immunogenicity over time remains largely unknown. To support the development of an optimal treatment protocol, we studied the impact of prolonged BRAFi exposure on the recognition of melanoma cells by T cells in different patient models. We demonstrate that autologous CD8+ tumor-infiltrating lymphocytes (TILs) efficiently recognized short-term (3, 7 days) BRAFi-treated melanoma cells but were less responsive towards long-term (14, 21 days) exposed tumor cells. Those long-term BRAFi-treated melanoma cells showed a non-proliferative dedifferentiated phenotype and were less sensitive to four out of five CD8+ T cell clones, present in the preexisting TIL repertoire, of which three recognized shared antigens (Tyrosinase, Melan-A and CSPG4) and one being neoantigen-specific. Only a second neoantigen was steadily recognized independent of treatment duration. Notably, in all cases the impaired T cell activation was due to a time-dependent downregulation of their respective target antigens. Moreover, combinatorial treatment of melanoma cells with BRAFi and an inhibitor of its downstream kinase MEK had similar effects on T cell recognition. In summary, MAP kinase inhibitors (MAPKi) strongly alter the tumor antigen expression profile over time, favoring evolution of melanoma variants cross-resistant to both T cells and MAPKi. Our data suggest that simultaneous treatment with MAPKi and immunotherapy could be most effective for tumor elimination.