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Project management is sufficiently described in the source literature. However, numerous published scientific works refer mainly to examples of good practice in project management in business entities, and unfortunately higher education establishments (HEE) are marginally analysed. Therefore, the analyses defined in study aim to evaluate the performance indicators of project management in universities, on the example of Gdynia Maritime University. In the studies the research in action method was applied as well as the technique of direct interview with project or department managers involved in the project implementation. The analyses covered selected projects that were implemented at the University between 2017 and 2021. Moreover, research questions were formulated, i.e., 1) Which indicators are used to assess the efficiency of project management at HEE?, 2) Does the surveyed HEE analyse the project management efficiency assessment? In order to conduct the study, the Author’s indicators were proposed to assess the efficiency of process management during the implementation of projects in HEE and these were applied in the analysis presented in this article. It was found that the surveyed university does not apply indicators to assess the efficiency of project management. Whereas the usefulness of indicators proposed by the Authors to study the efficiency of project management was confirmed by calculations performed. On these grounds, the Authors recommend the University authorities should consider implementing these indicators.

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19− K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA–specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467–80. ©2018 AACR.

Supplementary Video 1. Time-lapse sequence of a serial killer NK cell attacking a cluster of six 221 cells in a 50Ã-50Ã-300 μm3 microwell. The movie plays the same sequence simultaneously side-by-side in fluorescence (left) and bright field (right). The NK cell is shown in blue (in both panels), live targets are shown in green and dead targets in red. At the end of the movie five target cells have been killed. Total imaging time was 4.5 hours with 10 minutes between frames.

Supplemental Table 1

Supplemental Materials

The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase–associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition–induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies.Significance:These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.

Supplementary materials and methods

Supplementary Figures 1-6. Suppl. Fig. 1. Mel-ILP induces release of certain cytokines, however does not affect infiltration of immune cells into tumor bed. Suppl. Fig. 2. Melphalan induces apoptosis of melanoma cells. Suppl. Fig. 3 Melphalan induces exposure of danger signals. Suppl. Fig. 4. Dying human melanoma cells induce danger signaling-independent in vitro stimulation of immune cells. Suppl. Fig. 5. In vitro treated melanoma cells do not strongly activate NK cells. Suppl. Fig. 6. (A) The phenotypic maturation of murine iDCs (B) Effect of antibodybased elimination of CD4+ and CD8+ cells in prophylactic mice vaccination experiment

Supplementary Tables 1-3. Supplementary Table 1. Meta-data analysis of clinical response to melphalan-based isolated limbtreatment procedures in melanoma patients. Supplementary Table 2. Meta-data analysis of clinical response to melphalan/TNF or Mel/TNF/IFNγ combination therapies for isolated limb-treatment procedures in melanoma patients. Supplementary Table 3. Follow up information about the patients whose tumour samples were used for analysis of infiltration of immune cells and expression levels of cytokines.

Systemic chemotherapy generally has been considered immunosuppressive, but it has become evident that certain chemotherapeutic drugs elicit immunogenic danger signals in dying cancer cells that can incite protective antitumor immunity. In this study, we investigated whether locoregionally applied therapies, such as melphalan, used in limb perfusion for melanoma (Mel-ILP) produce related immunogenic effects. In human melanoma biopsies, Mel-ILP treatment upregulated IL1B, IL8, and IL6 associated with their release in patients' locoregional sera. Although induction of apoptosis in melanoma cells by melphalan in vitro did not elicit threshold levels of endoplasmic reticulum and reactive oxygen species stress associated with danger signals, such as induction of cell-surface calreticulin, prophylactic immunization and T-cell depletion experiments showed that melphalan administration in vivo could stimulate a CD8+ T cell–dependent protective antitumor response. Interestingly, the vaccination effect was potentiated in combination with exogenous calreticulin, but not tumor necrosis factor, a cytokine often combined with Mel-ILP. Our results illustrate how melphalan triggers inflammatory cell death that can be leveraged by immunomodulators such as the danger signal calreticulin. Cancer Res; 75(8); 1603–14. ©2015 AACR.

The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase–associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition–induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. Significance: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.

Oxidative stress, caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses, is one of the characteristic features of cancer. Here, we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo, when compared with blood or normal subcutaneous fluid. Therefore, we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells, and by comparing T, B, and NK cells' sensitivities to redox stress and their antioxidant capacities, we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However, the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore, we genetically modified NK cells to stably overexpress PRDX1, which led to increased survival and NK cell activity in redox stress conditions. Finally, we generated PD-L1–CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide, at concentrations detected in the TME, suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors.

Histone deacetylase (HDAC) inhibitors, approved for the treatment of cutaneous T-cell lymphoma (CTCL), are non-selective agents associated with an unsatisfactory response and considerable side-effects. Targeting single HDAC isoforms is considered to provide novel therapeutic options. HDAC6 is overexpressed in primary samples from patients with CTCL and preclinical studies using transgenic mice that spontaneously develop a CTCL-like disease, have suggested that combinations including HDAC6 inhibitors may be successful in the treatment of CTCL. PI3K inhibition is currently being tested in clinical trials for CTCL with promising results. Since HDAC6 is known to diminish the activity of Akt via its deacetylation, the aim of the present study was to evaluate the therapeutic potential of selective HDAC6 inhibitors in combination with PI3K inhibitors in CTCL. Through the genetic and pharmacological inhibition of HDAC6, it was demonstrated that combining HDAC6 with PI3K inhibition may be an attractive therapeutic option for patients with CTCL.

BackgroundImmune checkpoint inhibitors and chimeric antigen receptor (CAR)-based therapies have transformed cancer treatment. Recently, combining these approaches into a strategy of PD-L1-targeted CAR has been proposed to target PD-L1high tumors. Our study provides new information on the efficacy of such an approach against PD-L1low targets.MethodsNew atezolizumab-based PD-L1-targeted CAR was generated and introduced into T, NK, or NK-92 cells. Breast cancer MDA-MB-231 and MCF-7 cell lines or non-malignant cells (HEK293T, HMEC, MCF-10A, or BM-MSC) were used as targets to assess the reactivity or cytotoxic activity of the PD-L1–CAR-bearing immune effector cells. Stimulation with IFNγ or with supernatants from activated CAR T cells were used to induce upregulation of PD-L1 molecule expression on the target cells. HER2–CAR T cells were used for combination with PD-L1–CAR T cells against MCF-7 cells.ResultsPD-L1–CAR effector cells responded vigorously with degranulation and cytokine production to PD-L1high MDA-MB-231 cells, but not to PD-L1low MCF-7 cells. However, in long-term killing assays, both MDA-MB-231 and MCF-7 cells were eliminated by the PD-L1–CAR cells, although with a delay in the case of PD-L1low MCF-7 cells. Notably, the coculture of MCF-7 cells with activated PD-L1–CAR cells led to bystander induction of PD-L1 expression on MCF-7 cells and to the unique self-amplifying effect of the PD-L1–CAR cells. Accordingly, PD-L1–CAR T cells were active not only against MDA-MD-231 and MCF-7-PD-L1 but also against MCF-7-pLVX cells in tumor xenograft models. Importantly, we have also observed potent cytotoxic effects of PD-L1–CAR cells against non-malignant MCF-10A, HMEC, and BM-MSC cells, but not against HEK293T cells that initially did not express PD-L1 and were unresponsive to the stimulation . Finally, we have observed that HER-2–CAR T cells stimulate PD-L1 expression on MCF-7 cells and therefore accelerate the functionality of PD-L1–CAR T cells when used in combination.ConclusionsIn summary, our studies show that CAR-effector cells trigger the expression of PD-L1 on target cells, which in case of PD-L1–CAR results in the unique self-amplification phenomenon. This self-amplifying effect could be responsible for the enhanced cytotoxicity of PD-L1–CAR T cells against both malignant and non-malignant cells and implies extensive caution in introducing PD-L1–CAR strategy into clinical studies.

Despite the introduction of a plethora of different anti-neoplastic approaches including standard chemotherapy, molecularly targeted small-molecule inhibitors, monoclonal antibodies, and finally hematopoietic stem cell transplantation (HSCT), there is still a need for novel therapeutic options with the potential to cure hematological malignancies. Although nowadays HSCT already offers a curative effect, its implementation is largely limited by the age and frailty of the patient. Moreover, its efficacy in combating the malignancy with graft-versus-tumor effect frequently coexists with undesirable graft-versus-host disease (GvHD). Therefore, it seems that cell-based adoptive immunotherapies may constitute optimal strategies to be successfully incorporated into the standard therapeutic protocols. Thus, modern cell-based immunotherapy may finally represent the long-awaited “magic bullet” against cancer. However, enhancing the safety and efficacy of this treatment regimen still presents many challenges. In this review, we summarize the up-to-date state of the art concerning the use of CAR-T cells and NK-cell-based immunotherapies in hemato-oncology, identify possible obstacles, and delineate further perspectives.

Immunooncology gained great attention in cancer treatment over the last few years. Adoptive cell therapy is a type of immunotherapy that uses cytotoxic lymphocytes or NK cells. NK cells precisely recognize and kill cancer cells without affecting healthy cells. These properties make NK cells an attractive strategy with potential therapeutic applications. NK cells can be further modified with chimeric antigen receptors (CAR). Currently, several dozen clinical trials with NK cells are registered for cancer treatment, including several CAR-NK cells or NK-92 cell line modified with CAR. Despite the undeniable success of immunotherapy in recent years, many concerns still remain unresolved. One of the challenges for the effective immunotherapy is the immunosuppressive tumor environment. Numerous studies indicate that persistent oxidative stress, an important element associated with cancer, inhibits NK cell activity. However, the exact mechanisms responsible for protecting NK cells against suppressive oxidative stress are not identified. Therefore, in my doctoral dissertation co-financed by the Scientific Polpharma Foundation doctoral scholarship I focused on investigating and identifying mechanisms protecting NK cells against oxidative stress. The obtained results indicate that NK cells are the most sensitive to the oxidative stress, when compared withother immune cells. Moreover, inhibition of PRDX1-TXN1-TXNRD1 system impairs NK cell effector functions, while the increase of PRDX1 significantly improves the survival of NK cells and CAR-NK cells in the presence of H2O2, resulting in improved effectiveness of NK cells under the oxidative stress conditions.

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogeneous blood cancer characterized by abnormal expansion of immature B cells. Although intensive chemotherapy provides high cure rates in a majority of patients, subtypes harboring certain genetic lesions, such as MLL rearrangements or BCR-ABL1 fusion, remain clinically challenging, necessitating a search for other therapeutic approaches. Herein, we aimed to validate antioxidant enzymes of the thioredoxin system as potential therapeutic targets in BCP-ALL. We observed oxidative stress along with aberrant expression of the enzymes associated with the activity of thioredoxin antioxidant system in BCP-ALL cells. Moreover, we found that auranofin and adenanthin, inhibitors of the thioredoxin system antioxidant enzymes, effectively kill BCP-ALL cell lines and pediatric and adult BCP-ALL primary cells, including primary cells co-cultured with bone marrow-derived stem cells. Furthermore, auranofin delayed the progression of leukemia in MLL-rearranged patient-derived xenograft model and prolonged the survival of leukemic NSG mice. Our results unveil the thioredoxin system as a novel target for BCP-ALL therapy, and indicate that further studies assessing the anticancer efficacy of combinations of thioredoxin system inhibitors with conventional anti BCP-ALL drugs should be continued.

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy caused by accumulation of immature B cells. Advances in the field, especially in genomic studies and new treatment development, significantly improved the prognosis of B-ALL over the last decades. However, the B-ALL subtype harboring Philadelphia chromosome t(9;22)(q34;q11) (Ph+ B-ALL) still remains a therapeutic challenge despite available targeted treatment with tyrosine kinase inhibitors (TKIs). The novel therapeutic regimens for adult B-ALL often include rituximab (RTX) - a monoclonal anti-CD20 antibody - for patients with >20% CD20+ blasts. Moreover, drug combinations including approved TKIs and antibodies are being tested in clinical trials. Since some TKIs display immune effector cell inhibition, investigating their immunomodulatory effects in the context of combined chemo-immunotherapies is clinically relevant for the rational combination design. We found that blasts isolated from Ph+ B-ALL patients showed the highest CD20 expression on mRNA level among defined B-ALL genetic subtypes. As Ph+ CD20+ patients may be qualified for treatment with TKIs along with RTX, this result prompted the need of investigating the effects of TKIs on immune effectors. We therefore compared how first-, second- and third-generation TKIs (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) and allosteric inhibitor asciminib recently approved for the treatment of chronic myeloid leukemia (CML) affect innate and antibody-mediated cytotoxicity. B-ALL cell lines, patient-derived lymphoblasts propagated in NSG mice (primografts) and ex vivo whole blood assays were used for testing the efficacy of effector cell-mediated mechanisms in the presence of TKIs. The TKIs were compared in NK-cell based natural cytotoxicity tests, antibody-dependent cytotoxicity (ADCC) tests and phagocytosis assays with monocyte-derived macrophages. Dasatinib was also tested using whole blood ex vivo ADCC assays before and after administration of the TKI. Out of six tested TKIs, asciminib presented the most favorable profile, causing slight or no effector cell inhibition in both innate and antibody-mediated responses. In contrast, dasatinib significantly decreased the immune cells activity in all tests, reducing natural and RTX-mediated cytotoxicity of NK cells against B-ALL blasts. Dasatinib and ponatinib significantly inhibited in vitro phagocytosis of primograft B-ALL cells. To a lesser extent, cytotoxicity and phagocytosis were suppressed by imatinib, bosutinib and nilotinib. Importantly, we confirmed that the orally administered dasatinib impairs the NK cell activity in patients’ blood. Our findings indicate that the TKIs differentially impact RTX-mediated effector mechanisms. The novel, allosteric TKI asciminib could be preferentially used in combination with RTX-based immunotherapy. This work was supported by the Polish National Science Centre grant 2019/35/B/NZ5/01428 and by the Ministry of Education and Science within “Regional Initiative of Excellence” program 013/RID/2018/19." Citation Format: Krzysztof Domka, Martyna Poprzeczko, Zuzanna Urbanska, Lukasz Komorowski, Agata Pastorczak, Klaudyna Fidyt, Agnieszka Dabkowska, Mieszko Lachota, Magdalena Winiarska, Karolina Siudakowska, Elzbieta Patkowska, Łukasz Sędek, Bartosz Perkowski, Beata Krzymieniewska, Malgorzata Firczuk. Optimizing the therapeutic potential of tyrosine kinase inhibitors in chemo-immunotherapy of B-cell acute lymphoblastic leukemia involving rituximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3324.

The prognosis for B-cell precursor acute lymphoblastic leukemia patients with Mixed-Lineage Leukemia (MLL) gene rearrangements (MLLr BCP-ALL) is still extremely poor. Inhibition of anti-apoptotic protein BCL-2 with venetoclax emerged as a promising strategy for this subtype of BCP-ALL, however, lack of sufficient responses in preclinical models and the possibility of developing resistance exclude using venetoclax as monotherapy. Herein, we aimed to uncover potential mechanisms responsible for limited venetoclax activity in MLLr BCP-ALL and to identify drugs that could be used in combination therapy. Using RNA-seq, we observed that long-term exposure to venetoclax in vivo in a patient-derived xenograft model leads to downregulation of several tumor protein 53 (TP53)-related genes. Interestingly, auranofin, a thioredoxin reductase inhibitor, sensitized MLLr BCP-ALL to venetoclax in various in vitro and in vivo models, independently of the p53 pathway functionality. Synergistic activity of these drugs resulted from auranofin-mediated upregulation of NOXA pro-apoptotic protein and potent induction of apoptotic cell death. More specifically, we observed that auranofin orchestrates upregulation of the NOXA-encoding gene Phorbol-12-Myristate-13-Acetate-Induced Protein 1 (PMAIP1) associated with chromatin remodeling and increased transcriptional accessibility. Altogether, these results present an efficacious drug combination that could be considered for the treatment of MLLr BCP-ALL patients, including those with TP53 mutations.

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19− K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA–specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467–80. ©2018 AACR.

Background: Venetoclax (VEN), a specific BCL2 inhibitor, exerts anti-leukemic effects in various high-risk (HR) B-ALL subtypes, such as ALL with mixed lineage leukemia (MLL) gene rearrangements (MLLr ALL) (PMID: 26711339), Philadelphia chromosome-positive (Ph+) (PMID 30546081) or hypodiploid B-ALL (PMID 30862722). Nevertheless, despite high rationale for targeting BCL2 in these subtypes of B-ALL, VEN monotherapy is not effective enough to completely eliminate leukemic cells. For this reason identification of other drugs that could sensitize leukemic cells to VEN may become beneficial treatment strategy in HR ALL. Previously, we showed that the enzymes of the thioredoxin system are upregulated in primary B-ALL cells and that auranofin (AUR), a thioredoxin reductase inhibitor, effectively kills leukemic cells in vitro and in vivo. Importantly, elements of the thioredoxin system are not only balancing redox homeostasis within the cells, but may also interact with other pathways, including anti-apoptotic signaling. Considering above, we hypothesized that AUR may potentiate VEN efficacy in HR B-ALL. Methods: To evaluate cytostatic/cytotoxic effects of VEN+AUR combination by MTT assay and propidium iodide (PI)-staining we used HR B-ALL cell lines, including SEM (MLLr ALL), BV-173 (Ph+ ALL) and NALM-16 (hypodiploid ALL). Patient derived xenograft cells (PDX) were generated through long-term propagation of primary B-ALL samples in immune-deficient NSG mice. Ex vivo drug testing in co-culture system was performed using primary bone marrow-derived mesenchymal stem cells (BM-MSC) and murine stromal OP9 cell line. NOXA genomic knockout (KO) in SEM cells was established by CRISPR/Cas9 system. Chromatin accessibility within PMAIP1 gene (encodes for NOXA) was detected using ATAC-seq. Results: We observed that AUR sensitizes HR B-ALL cell lines to VEN, as determined by MTT and PI-staining. Further, we mimicked the bone marrow support of stromal cells towards B-ALL and evaluated its impact on the response to VEN+AUR. For this reason we employed an ex vivo co-culture system of B-ALL PDX cells with primary BM-MSC or an OP9 cell line. In all tested PDX samples representing diagnostic/relapsed MLLr ALL (n=8), Ph+ ALL (n=2) and Ph-like ALL (n=2) we observed synergistic effect of this combination (Fig. 1A). Next, we determined the efficacy of VEN+AUR combination in vivo using a PDX model of MLLr B-ALL. We observed that administration of VEN+AUR diminished the progression of leukemia during a 3 week-long treatment more effectively than any single drug alone, which reflected in longer survival of NSG mice (Fig. 1B). Subsequently, we aimed to uncover the mechanism responsible for the synergistic action of VEN+AUR. In cells treated with both drugs we observed enhanced caspase activation and changes in the levels of BCL2 family proteins involved in apoptotic signaling. In particular, we found that AUR strongly upregulates a pro-apoptotic NOXA protein, both in HR B-ALL cell lines and in MLLr ALL PDX samples (Fig. 1C). To evaluate whether NOXA induction is functionally relevant for the cell death mediated by VEN+AUR, we generated SEM cells with a NOXA genomic KO. Lack of NOXA significantly abolished VEN-single agent as well as VEN+AUR combination cytotoxicity, demonstrating its dependence on NOXA expression (Fig. 1D). We then showed that NOXA is regulated at the transcriptional level, as co-treatment with AUR and the transcription inhibitor, actinomycin D, abolished AUR-mediated NOXA induction at mRNA and protein levels in SEM cells. Additionally, to test whether AUR-treatment itself provokes changes in chromatin accessibility within the NOXA encoding gene (PMAIP1) we performed ATAC-seq. We observed a clear increase in accessibility at PMAIP1 in response to AUR, which correlated with transcriptional induction of NOXA. Moreover, ChIP-qPCR revealed that increased ATAC peaks within PMAIP1 were associated with an increase in H3 lysine 27 acetylation (H3K27ac) - an epigenetic mark associated with open chromatin conformation. Conclusions: Our results demonstrate that FDA-approved drug, AUR, is a promising candidate to be used in combination with VEN for the therapy of HR B-ALL subtypes. Importantly, NOXA induction by AUR plays a central role in the VEN+AUR synergistic cytotoxicity. More studies elucidating the mechanism of NOXA upregulation by AUR are underway. Disclosures Milne: OxStem Oncology (OSO), a subsidiary company of OxStem Ltd.: Other: Founding shareholder .

Natural Killer (NK) cells play an essential role in cancer surveillance and have a unique capability of spontaneous cytotoxicity against cancer cells. The human NK cell repertoire is functionally diversified through a tightly regulated differentiation process characterized by an early transition from CD56bright to CD56dim NK cells, followed by coordinated changes in expression of inhibitory receptors, including NKG2A and killer cell immunoglobulin-like receptors (KIR). The acquisition of self HLA class I binding KIRs during NK cell differentiation tunes the cytotoxic potential of NK cells in a process termed education, characterized by increased loading of granzyme B in dense core granules. Although NK cell differentiation and education are critical determinants of the functional potential of the cell, little is known about how these events shape the migratory behavior of NK cells. To mediate appropriate and directed immune response against cancer, NK cells must be capable of migration to the tumor site. This process is mediated by chemokines, which guide cell migration by binding to their specific receptors. For example, in multiple myeloma, CXCR3 and CCR5 ligands (MIG, IP-10, and MIP-1a) are significantly upregulated in the bone marrow compared to healthy controls, affecting the composition of immune cells in the tumor microenvironment. In order to delineate the homing patterns of distinct NK cell subsets, we used high-dimensional flow cytometry combined with functional assays to map the NK cell chemokine receptor expression and migratory behavior. We screened resting and cytokine/feeder cell stimulated peripheral blood NK cells for the expression of a panel of 20 chemokine receptors (A). Based on CD56, CD57, NKG2A, and KIR expression, NK cells were divided into 6 phenotypically and functionally distinct subsets that were ordered according to their differentiation status (B). We found that the expression of CX3CR1, CXCR1, CXCR2, and CMKLR1 gradually increased during differentiation, whereas the expression of CXCR3, CCR7, and CCR5 was lower in more differentiated NK cells. CXCR4, CCR4, and CCR2 expression was relatively uniform across all subsets. Interestingly, CCR1 and CXCR6 were expressed mainly on less differentiated NKG2A+ CD56dim NK cells (B). Next, we stratified the chemokine receptor expression on mature KIR+ NK cells based on the expression of self (educated) or non-self KIR (uneducated). Educated NK cells expressed CXCR1, CX3CR1, CCR5, and CMKLR1 at higher levels than the uneducated NK cells. Conversely, CXCR3 was expressed at lower levels on educated NK cells (C). No difference was observed for CXCR2 expression. To determine whether the observed differences in chemokine receptor expression translate into altered chemokine responsiveness between the subsets, we combined the transwell system with multicolor flow cytometry. We found that the chemokine-induced migration capability of NK cells correlated closely with the expression level of corresponding chemokine receptor, leading to subset specific responses to various chemokine gradients (D). The present results show that peripheral blood NK cell chemokine receptor profile changes in a coordinated fashion during NK cell differentiation and is further influenced by the expression of self-specific KIR. Interestingly, receptors which expression declines during NK cell differentiation (CCR5, CCR7, and CXCR3) are commonly associated with adaptive T cell responses to viruses, whereas receptors that are upregulated along the differentiation axis (CXCR1, CXCR2, CX3CR1, CMKLR1) are typical for neutrophils and macrophages as a part of the innate immune response. Thus, our results suggest that NK cell differentiation and education processes together shape the NK cell migratory capabilities to promote homing of the most functional NK cell subsets to the site of inflammation and serve as the first line of defense in the immune response to pathogens and tumors. Figure Disclosures Malmberg: Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.