CTCL Cell Research Articles

Combination of JAKi and HDACi Exerts Antiangiogenic Potential in Cutaneous T-Cell Lymphoma.

Angiogenesis plays a pivotal role in the growth and metastasis of tumors, including the development and progression of cutaneous lymphomas. The chick embryo CAM model has been utilized in various studies to assess the growth rate, angiogenic potential, and metastatic capability of different tumor types and malignant cell lines. However, the precise mechanisms of angiogenesis in CTCL and the influence of Ruxolitinib or Resminostat on angiogenesis in hematological malignancies and solid tumors are not well understood. Recent in vitro and in vivo data have demonstrated the synergistic inhibition of tumorigenesis and metastasis in experimental models of CTCL when using the combination of Resminostat (HDACi) with Ruxolitinib (JAKi). The present work aims to elucidate the effects of this combination on the tumor microenvironment's vascular components. We investigated the effects of Ruxolitinib (JAKi) in combination with Resminostat (HDACi) treatment in transendothelial migration of CTCL cells (106 MyLa and SeAx) by using a transwell-based transendothelial migration assay and tumor angiogenesis in vivo. We used the CTCL chick embryo CAM model with xenografted tumors derived from implanted MyLa and SeAx cells and administered topically 15 μM ruxolitinib and 5 μM Resminostat every two days during a 5-day period. JAKi and HDACi inhibited CTCL cell transendothelial migration by 75% and 82% (p < 0.05) in both CTCL engrafted cells (MyLa and SeAx, respectively) compared to the untreated group. Moreover, the combination of ruxolitinib with resminostat blocked angiogenesis by significantly reducing the number of blood vessel formation by 49% and 34% in both MyLa and SeAx, respectively (p < 0.05), indicating that the proposed combination exerted significant anti-angiogenic effects in the CAM CTCL model. Overall, these data provide valuable insights into potential therapeutic strategies targeting angiogenesis in CTCL, paving the way for more effective treatment approaches in the future.

Evaluation of Sézary cell marker expression and cell death behaviour upon invitro treatment by flow cytometry in Sézary syndrome patients.

The diagnosis of Sézary syndrome (SS) relies on the identification of blood Sézary cells (SC) by different markers via flow cytometry. Treatment of SS is challenging since its pathogenesis is characterized by cell death resistance rather than hyperproliferation. In this study, we establish an integrated approach that considers both the expression of SC markers and sensitivity to cell death both spontaneously and upon invitro treatment. Peripheral blood mononuclear cells were isolated from 20 SS patients and analysed for the SC markers CD7 and CD26 loss as well as CD158k and PD1 gain. The cells were then treated with different established and experimental therapies invitro and cell death was measured. Spontaneous and therapeutically induced cell death were measured and correlated to cellular marker profiles. In the marker-positive cells, spontaneous cell death sensitivity was reduced. Different treatments invitro managed to specifically induce cell death in the putative CTCL cell populations. Interestingly, a repeated analysis after 3 months of treatment revealed the CTCL cell death sensitivity to be restored by therapy. We propose this novel integrated approach comprising the evaluation of SC marker expression and analysis of cell death sensitivity upon treatment that can also enable a better therapy stratification.

Abstract 2907: Therapeutic targeting miR-146a and miR-21 induce malignant cell death and regulate CD8+ T-cell function in mycosis fungoides with large-cell transformation

Abstract Large cell transformation of mycosis fungoides (LCT-MF) occurs in 20-50% of advanced MF and is associated with an aggressive clinical course and poor survival not to overcome by any standard treatment regimen. We have previously identified a distinct miRNA expression profile in LCT-MF from that of non-transformed MF with significant upregulation of miR-21 and miR-146a. Our analyses demonstrated the involvement of genes for immune checkpoint pathways such as ICOS-ICOSL and PD1-PDL1 signaling (Di Raimondo C et al. Cancers 2021). Here, we aimed to investigate the efficacy of antagomiR-146a and -21 (amiR-146a, and -21) on the tumor growth and CD8+ tumor infiltrating lymphocyte exhaustion in LCT-MF. The amiR-146a, and -21 were synthesized in our DNA/RNA Synthesis Core by linking CpG-D19. In vitro, CTCL cell lines (Myla and HH) were treated with amiR-146a and -21, the cell viability was assessed by the 2,5-diphenyl-2H-tetrazolium bromide assay and cell apoptosis was evaluated using apoptosis assay. We found that amiR-146a, and -21 synergistically inhibited the proliferation of MyLa and HH cells due to the activation of apoptosis through Caspase3/7 pathways and inducing cell cycle arrest by blocking STAT3/CDK1/Cyclin B1 pathway. Our RNA-seq data indicated that the exhausted CD8+ T cells express elevated amounts of STAT3, IRF4, and BATF in LCT-MF compared with non-LCT MF. To evaluate the functional importance of amiR-146a, and -21 on CD8+ T cell exhaustion, we induced an exhausted state of CD8+ T cells with high level of immune checkpoints and dysfunctional cytokine production by continues anti-CD3/DC28 beads and culture supernatant (MyLa or HH cell) exposure. Our data revealed that amiR-146a and -21 attenuated the CD8+ T cell exhaustion by blockade of immune checkpoints and STAT3/IRF4/BATF pathway to trigger the cytotoxic immune response. Collectively, the findings of our study suggest that targeting miR-146a and -21 is a promising and novel therapeutic strategy for LCT-MF. Citation Format: Zhen Han, Piotr Swiderski, Xiwei Wu, Yate-Ching Yuan, Jun Wu, Chingyu Su, Hanjun Qin, Steven Rosen, Christiane Querfeld. Therapeutic targeting miR-146a and miR-21 induce malignant cell death and regulate CD8+ T-cell function in mycosis fungoides with large-cell transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2907.

Abstract 5720: Targeting CDK9 in cutaneous T-cell lymphoma using patient-derived xenograft models

Abstract Cutaneous T-cell lymphoma (CTCL) is a rare form of non-Hodgkin lymphoma. Mycosis fungoides and Sézary syndrome (MF/SS) are the most common subtypes in CTCL. Early-stage MF/SS has a favorable prognosis. The advanced-stage CTCL has limited treatment options and a median survival of &amp;lt;5 years, highlighting the urgent need to develop targeted therapies in CTCL. Preclinical studies of CDK9 inhibitors have shown anti-tumor activity in various subtypes of hematologic malignancies. Several CDK9 inhibitors have entered clinical trials for lymphomas and leukemias. However, whether targeting CDK9 in CTCL is a therapeutic option is little known and has resulted in CTCL not being included in ongoing clinical investigations of CDK9 inhibitors. AZD4573 is a highly selective and potent CDK9 inhibitor currently under clinical investigation for Hodgkin and non-Hodgkin lymphomas. It was shown that AZD4573 induces tumor cell death through depletion of MCL-1. Overexpression of MCL-1 has been shown in CTCL cell lines and skin biopsy specimens of CTCL patients. MCL-1 expression appears to be particularly elevated in patients with advanced stage disease. Here, we investigated the ex vivo anti-tumor activity of AZD4573 using patient-derived CTCL xenograft models (PDXs). Primary tumor cells harvested from CTCL PDX mice were activated with CD2/CD3/CD28 beads and incubated in hIL-2 and hIL-7 at an optimal cell density. Subsequently, cells were exposed to AZD4573 at various concentrations for 8 hr, then treated cells were harvested, washed, and placed in fresh media for another 48 hr. Growth inhibition was measured by the Real-Time Glo cell viability assay, and apoptotic activity was assessed by flow cytometry analysis of Caspase 3/7 activity and Annexin V + propidium iodide (PI) double staining. Additionally, we conducted RNA-seq analysis for MCL-1 expression in CTCL PDX primary tumor cells. AZD4573 inhibited cell proliferation in a dose-dependent manner in primary tumor cells from three CTCL PDX mice (PRS-1, PRS-2 PRS-3), with GI50 of 43.5 nM, 175.4 nM, and 113.5 nM, respectively. Furthermore, AZD4573 significantly increased caspase 3/7 activity and apoptosis in PRS-1 and PRS-3, but not PRS-2 primary tumor cells. In addition, our RNA-seq analysis showed that compared with normal controls (normal PBMC from healthy donors), the MCL-1 gene expression is increased 10-fold (P-value &amp;lt;0.0001) in PRS1 cells. The MCL-1 gene expression in PRS2 and PRS3 cells is the same as normal control. Furthermore, PRS-1, which has the highest MCL-1 expression level, is most sensitive to AZD4573. In conclusion, AZD4573 demonstrated dose-dependent growth inhibition and apoptosis induction in primary CTCL tumor cells. In addition, the expression level of MCL-1 appeared to be correlated with the ex vivo anti-tumor activity of AZD4573. Our findings provide a rationale for clinical investigation of targeting CDK9 in CTCL. Citation Format: Yan-Jin Liu, Chi-Heng Wu, Laura Pincus, Weiyun Z. Ai. Targeting CDK9 in cutaneous T-cell lymphoma using patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5720.

Abstract 4605: AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-cell lymphoma models

Abstract Peripheral T-cell Lymphoma (pTCL) makes up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor prognosis. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E. Here we examine the efficacy of CDK9 inhibition for the treatment of pTCL. CDK9 regulates transcription through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor; acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are often overexpressed in hematologic tumors. It has been shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression of BCL-2 family members is highly heterogeneous in pTCL, with MCL-1 being the most universally expressed. Using MCL-1 inhibitor AZD5991, we showed statistically significant benefit in survival when combined with CHOP in MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition, we have previously reported the importance of the anti-apoptotic protein BFL-1 in cell survival in NHL, including a subset of pTCL (Boiko et al 2021). AZD4573 treatment showed a range of activity across the panel of 18 human pTCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, with EC50 values less than 100 nM, and 13 reached a max caspase activation of over 40%. pTCL subtypes that responded to AZD4573 treatment included ALCL, NK-TCL, and PTCL-NOS and CTCL. Acute AZD4573 treatment resulted in decreased pSer2-RNAP2 and reduced protein levels of c-MYC and MCL-1. To determine what was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 for viability after KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that efficacy is mediated through MCL-1 in ALCL pTCL cell lines assessed. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and measured viability after 24-72 hours using RealTime-Glo after 24hr exposure to CHOP with AZD4573 added for the last 6hrs. Adding AZD4573 to CHOP in vitro increased the efficacy in pTCL ALCL cell lines with MCL-1 dependency, but not in CTCL cell line with BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels but not MCL-1, suggesting that combination benefit may be driven through c-MYC. This data suggests that treatment with AZD4573 as a monotherapy or in combination with CHOP regimen would be an effective therapeutic strategy in pTCL. Citation Format: Danielle Sinead Potter, India Ott, Jamal Saeh, Richard Olsson, Stephen Fawell, Lisa Drew, Justine Roderick. AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4605.

GATA-3 Dependent Gene Transcription Is Impaired upon HDAC Inhibition

Background: GATA-3 is a lymphoid-lineage specific zinc-finger transcription factor that plays a pivotal role in the development and homeostatic survival of thymic T-cell progenitors and subsets of their mature progeny. Diverse T-cell lymphomas (TCL), derived from conventional (non-malignant) T cells expressing GATA-3, highly express GATA-3 and a GATA-3 dependent transcriptional program, and are generally associated with poor outcomes. Upon binding gene enhancers and promoters, GATA-3 regulates gene transcription, including those with cell-autonomous (e.g. oncogenes, including c-Myc) and non-cell-autonomous (e.g. type 2 cytokines) functions in TCL. Therefore, GATA-3, and the transcriptional program it instigates, is a bona-fide proto-oncogene in these lymphomas. GATA-3 acetylation is dynamically regulated, and its acetylation controls its DNA binding affinity and target gene expression. Therefore, we sought to examine the extent to which HDAC inhibition may alter GATA-3 acetylation and functionally regulate its DNA binding capacity and target gene expression in CTCL. Methods: Integrated GATA-3 ChIP-seq and RNA-seq was performed in CTCL cell lines and primary specimens treated with clinically available HDACi. Complementary strategies were utilized to examine GATA-3/HDAC binding and GATA-3 acetylation in these cells. Results: In order to investigate early transcriptional changes upon HDACi treatment, a well characterized CTCL cell line (H9) and primary Sezary Syndrome (SS) specimens (n=3) were transcriptionally profiled. Gene set enrichment analysis (GSEA) was performed using the differentially expressed genes we identified, and a convergence on relevant T-cell growth and survival pathways was observed. GATA-3 target genes were differentially expressed upon HDACi treatment, and binding sites for GATA-3, and transcription factors (TF) it transcriptionally regulates, were enriched among HDACi responsive genes. Among the 5,510-6,811 transcripts that were differentially expressed upon either belinostat or romidepsin treatment, respectively, ≈6-7% of transcripts were GATA-3 targets. GATA-3 dependent transcripts were significantly altered upon treatment with belinostat or romidepsin in primary SS specimens. We next examined the association between GATA-3 and class I HDACs in CTCL cell lines. HDAC1/HDAC2 and GATA-3 binding was observed, suggesting that HDAC inhibition may lead to GATA-3 hyperacetylation. Treatment with the three clinically available HDACi led to a significant increase in GATA-3 acetylation. As GATA-3 acetylation regulates its ability to bind DNA and regulate transcription, we examined GATA-3 DNA binding using orthogonal approaches, including GATA-3 ChIP-seq, and a significant reduction in GATA-3 binding peaks was observed, culminating in significant changes in the GATA-3 dependent transcriptome. Conclusions: Collectively, these findings demonstrate that GATA-3 hyperacetylation upon HDAC inhibition significantly impairs GATA-3 DNA binding and GATA-3 dependent gene transcription. Impact: GATA-3 and its target genes identify clinically and transcriptionally distinct T-cell lymphomas, including CTCL, that are resistant to conventional chemotherapeutic agents and generally associated with dismal outcomes. Therefore, GATA-3 is a rational, albeit challenging, therapeutic target in these T-cell lymphomas. We have previously explored “upstream” strategies to target GATA-3 by exploiting transcription factors that regulate its expression, and have more recently explored “downstream” strategies by targeting its target genes, including ITK. To the best of our knowledge, the work described here is the first - and a clinically approved - strategy to therapeutically target GATA-3 directly, as HDACi significantly increase GATA-3 acetylation, and by doing so, impair its ability to bind DNA and regulate transcription of its gene targets. Consequently, these findings have significant implications for the interpretation of existing clinical trial datasets and for the development of future combinatorial strategies utilizing an HDACi-containing backbone in GATA-3 driven T-cell lymphomas.

First-in-Class HAT Activator (YF2) Combined with JAK/STAT Inhibitor (ruxolitinib) Unveils Potential Novel Treatment Approach for HDAC Inhibitor-Resistant CTCL

T-cell lymphomas are both aggressive and challenging to treat. This is in-part due to multiple epigenetic derangements found in T-cell lymphomas. One treatment strategy includes HDAC inhibitors; however, the duration of response is short for most patients which is consistent with many treatments for T-cell lymphoma. As a result, alternative treatments are required. YF2, a first-in-class histone acetyltransferase (HAT) activator, was invented at Columbia University. YF2 increases acetylation of histones by augmenting the activity of HAT enzymes. The JAK/STAT pathway contributes to CTCL lymphomagenesis through a number of cellular processes including cell cycle, proliferation, apoptosis, etc. JAKs activate STAT3, which activates the transcription of the anti-apoptotic Bcl-2-like protein, Bcl-XL. Additionally, HATs acetylate the pro-apoptotic p53 protein, increasing its stability and activity. Given the dependence on both the JAK/STAT pathway and epigenetic derangements in CTCL, we hypothesize that if the JAK inhibitor, ruxolitinib, induces apoptosis via modulation of Bcl-XL, and YF2 induces apoptosis via acetylation and activation of p53, then dual targeting of these complementary pathways could lead to synergistic apoptosis in CTCL. To study this hypothesis, we used two CTCL cell lines: HH [JAK wildtype (JAK WT)], and H9 [JAK1/3 gain-of-function mutated (JAK1/3 GOF)]. In addition, we generated a belinostat-resistant H9 cell line (H9-belino-R) by incrementally exposing H9 to increasing concentrations of belinostat. H9-belino-R resistance was observed at a 20-fold (IC 50 = 896.7nM, SEM ± 185.3) increase over that of the parental H9 (IC 50 = 43.8nM, SEM ± 4.61) ( figure 1). H9-belino-R retained significant resistance to other HDAC inhibitors such as romidepsin [(H9: IC 50 = 0.97nM (SEM ± 0.030), H9-belino-R: IC 50 = 1.38nM (SEM ± 0.028)] and panobinostat [H9: IC 50 = 3.11nM (SEM ± 0.19), H9-belino-R: IC 50 = 9.00nM (SEM ± 1.55)] as measured by the CellTiter-Glo Viability Assay. Interestingly, the IC 50 of the HAT activator, YF2, was retained between the resistant (14.84µM, SEM ± 0.46) and parental (12.45µM, SEM ± 0.27) cell lines ( figure 1). HH (JAK WT) showed significant cytotoxicity (63.5% live, 30.7% apoptotic, and 5.4% dead) after 4-day exposure to 10µM YF2 compared to limited cytotoxicity (93.3% live, 4.6% apoptotic, and 2.0% dead) in H9 (JAK GOF) as measured by flow cytometry with Annexin V. At baseline, active STAT3 (pSTAT3) was not detected in HH possibly contributing to its susceptibility to YF2 as the absence of pSTAT3 reduces the transcription Bcl-XL. As illustrated by figure 2, western blot analysis confirmed ruxolitinib-induced inhibition of active STAT3 (pSTAT3) with associated reduction in Bcl-XL expression in H9 and H9-belino-R. This may explain the observed synergistic cytotoxicity with YF2 and ruxolitinib in HH, H9 and H9-belino-R (Excess over Bliss scores: 4.85, 14.23, and 5.38 respectively) as determined by flow cytometry. In conclusion, the novel first-in-class HAT activator, YF2, retains activity in HDAC inhibitor-resistant CTCL. YF2's activity may be blunted by JAK1/3 GOF mutation; however, when combined with the JAK-inhibitor ruxolitinib, the response mirrored that of the HH (JAK WT) cell line. The combination of HAT activator and JAK inhibitor led to enhanced reduction of the anti-apoptotic protein Bcl-XL, potentially explaining synergistic apoptosis in CTCL. These preliminary findings provide us with evidence that suggests that the combination of YF2 and ruxolitinib can serve as a novel treatment combination for CTCL. Further study is planned to explore this treatment in murine models of disease.

AZD4573 in Combination with CHOP Increases Combination Benefit in Preclinical Peripheral T-Cell Lymphomas Models

Peripheral T-cell Lymphoma (pTCL) make up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor outcomes to standard of care therapy. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E and for CD30-postive pTCLs, brentuximab vedotin is approved. CDK9 regulates transcription elongation through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor, acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are frequently overexpressed in haematologic tumours. It has been previously shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression and dependencies of the anti-apoptotic BCL-2 family members is highly heterogeneous in pTCL; however MCL-1 is the most universally expressed BCL-2 family member. Using the MCL-1 inhibitor AZD5991, we have shown statistically significant benefit in survival when combined with CHOP in 2 MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition to MCL-1, we have previously reported the importance of the anti-apoptotic protein BFL-1 in mediating cell survival in NHL, including a subset of pTCL (Boiko et al 2021). Consistent with these findings, AZD4573 treatment exhibited a range of activity across the panel of 18 human TCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, having EC 50 values &amp;lt; 100 nM, and 13 reached a max caspase-3/7 activation of greater than 40%. pTCL cell lines that responded to AZD4573 treatment including ALCL, NK-TCL, and PTCL-NOS and CTCL subtypes. Acute AZD4573 treatment of pTCL cell lines resulted in decreased p-SerRNApoll II and reduced levels of c-MYC and MCL-1 protein levels. To determine if MCL-1 or c-MYC was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 after long term KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that AZD4573 efficacy is mediated through MCL-1, in ALCL pTCL cell lines tested. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and assessed for viability after 24-72 hours using RealTime-Glo after 18hr exposure to CHOP and AZD4573 added for the last 6hrs. Adding AZD4573 treatment to CHOP schedule in vitro increased the combination benefit in pTCL ALCL cell lines that show MCL-1 dependency, but not in CTCL cell line which show a BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels at 24-48 hours, but not MCL-1 suggesting that combination benefit may be driven through decreased c-MYC levels, as KO of c-MYC resulted is loss of cell viability in these models. These data suggested that treatment with AD4573 either as a monotherapy or in combination with CHOP, would be an effective therapeutic strategy in pTCL. AZD4573 monotherapy is currently being evaluated in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK in patients with relapsed/refractory pTCL.

In vitro evaluation of Neosetophomone B inducing apoptosis in cutaneous T cell lymphoma by targeting the FOXM1 signaling pathway

BackgroundCutaneous T cell lymphoma (CTCL) is a T cell-derived non-Hodgkin lymphoma primarily affecting the skin, with treatment posing a significant challenge and low survival rates. ObjectiveIn this study, we investigated the anti-cancer potential of Neosetophomone B (NSP-B), a fungal-derived secondary metabolite, on CTCL cell lines H9 and HH. MethodsCell viability was measured using Cell counting Kit-8 (CCK8) assays. Apoptosis was measured by annexin V/PI dual staining. Immunoblotting was performed to examine the expression of proteins. Applied Biosystems' high-resolution Human Transcriptome Array 2.0 was used to examine gene expression. ResultsNSP-B induced apoptosis in CTCL cells by activating mitochondrial signaling pathways and caspases. We observed downregulated expression of BUB1B, Aurora Kinases A and B, cyclin-dependent kinases (CDKs) 4 and 6, and polo-like kinase 1 (PLK1) in NSP-B treated cells, which was further corroborated by Western blot analysis. Notably, higher expression levels of these genes showed reduced overall and progression-free survival in the CTCL patient cohort. FOXM1 and BUB1B expression exhibited a dose-dependent reduction in NSP-B-treated CTCL cells.FOXM1 silencing decreased cell viability and increased apoptosis via BUB1B downregulation. Moreover, NSP-B suppressed FOXM1-regulated genes, such as Aurora Kinases A and B, CDKs 4 and 6, and PLK1. The combined treatment of Bortezomib and NSP-B showed greater efficacy in reducing CTCL cell viability and promoting apoptosis compared to either treatment alone. ConclusionOur findings suggest that targeting the FOXM1 pathway may provide a promising therapeutic strategy for CTCL management, with NSP-B offering significant potential as a novel treatment option.

HZ-R061, a Selective STAT3 Degrader with Activity in Preclinical Model of Hematologic Malignances

The Janus kinase/Signal transducer and activator of transcription (JAK-STAT) pathway is one of the core cancer pathways that integrates signals from cytokines, hormones and growth factors to induce or repress gene expression in cells . The pathway consists of four JAK kinases and seven STAT transcription factors (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6). STAT3 is the best-studied family member of the JAK-STAT pathway in cancer, and is a known oncogene in various types of tumor. So STAT3 is an attractive cancer therapeutic target, great efforts have been made to discover effective STAT3 inhibitor. However most of reported STAT3 inhibitors got failed for further development due to the low selectivity, weak binding affinity or poor drugability. STAT3 is considered as an undruggable target for long time. Recently, the proteolysis targeting chimeras (PROTAC) strategy has gained momentum for its promise in the discovery and development of an entirely new type of therapeutics for the treatment of human diseases. PROTAC molecule is a bifunctional small molecule designed to recruit cellular degradation machinery to induce targeted protein degradation in a catalytical way. PROTAC can be a new strategy to effectively target undruggable targets such as STAT3. HZ-R061 is a novel STAT3 selective degrader which is discovered and developed through our DaTProD® platform which was set up to promote PROTAC development. HZ-R061 can selective and effective degrade STAT3 in various types of cells including MOLM16, A549, SU-DHL1, karpas299, LY3, THP1 and human PBMC with DC50 values at nanomolar level. In in-vitro anti-proliferative assay, HZ-R061 exhibits potent anti-proliferative activity against CTCL and AML cell lines. Furthermore, MOLM16, SU-DHL1 and Karpas299 xenograft models were used for in-vivo anti-tumor evaluation. After IV administration of HZ-R061 (QW), the growth of tumor was significantly suppressed in all three models. As STAT3 is a master regulator in tumor microenvironment, inhibition of STAT3 could remodel TME towards in favor of anti-cancer. The combination of HZ-R061 with PD-(L)1 mAb was also conducted in CT26 model. HZ-R061 exhibits significantly synergistic effect in combination with PD-(L)1 mAb. Additionally, HZ-R061 also potently decreases phosphorylation level of STAT3 in IL-6 stimulated THP-1 cells with IC50 value of 3.8nM, which is 50 folds stronger than JAK inhibitor Ruxolitinib. HZ-R061 also shows strong effect in in-vitro Th17 differentiation assay and EAE model. These results suggest that HZ-R061 is also a promising drug candidate for various Th17 driven auto-immune diseases including MS, RA, SSc and IPF. In conclusion, we discovered a pre-clinical effective STAT3 degrader HZ-R061, which has potential for further development in tumor and auto-immune diseases.

P1278: ACTIVATION OF THE NECROPTOSIS PATHWAY PLAYS A ROLE IN SYNERGISM WITH COMBINATION OF THE IAP ANTAGONIST, TOLINAPANT AND HYPOMETHYLATING AGENTS (HMA) IN IN VITRO MODELS OF T-CELL LYMPHOMA (TCL).

Background: Peripheral T-cell lymphoma (PTCL) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas that often carry a poor prognosis with standard chemotherapy. Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In an ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma. HMAs have also shown clinical responses in some subsets of PTCL. Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. We have also shown that decitabine and tolinapant as single agent induced necroptosis in mouse TCL cell lines with the activation of the RIPK1/RIPK3/MLKL necroptosis pathway. In addition, re-expression of RIPK3 in CT26 cells by CRISPR increased lytic cell death on treatment with tolinapant indicating its essential role in the necroptosis pathway. Aims: We explored the sensitivity of a range of TCL lines to tolinapant, established the synergy coefficient and interrogated the role of necroptosis in the mechanism of synergy between tolinapant and drugs active against PTCL, the HMAs; azacytidine and decitabine Methods: A panel of 10 human TCL lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10 ng/ml of TNFa. For combination studies, each drug was tested at the IC10, IC20 and IC40 concentrations, in the presence or absence of TNFa. Synergy coefficient was calculated using the Excess over Bliss model with SynergyFinder. Additionally, the on-target effects of the drugs were measured by analyzing levels of the IAPs, DNMTs and key apoptosis and necroptosis markers, such as PARP and RIPK3 by Western blotting. Results: TCL Lines demonstrate varying sensitivities to tolinapant with the most sensitive cell line, ALK+ ALCL SUP-M2, having an IC50 as low as 20 nM ± 1 nM while a resistant CTCL cell line HH had an IC50 of over 20 µM. In combination experiments using both cell lines, tolinapant plus azacytidine or decitabine displayed a very high degree of synergism; Average synergy score of 50.9 and 49.8 with azacytidine and 22.6 and 31.3 with decitabine for HH and SUP-M2, respectively. Of note, a high degree of synergism was also seen with concentrations 10 fold lower of azacytidine or 100 fold lower of decitabine, added daily (Figure 1). The combination of tolinapant and the HMAs led to a decrease in the levels of cIAP1 and DNMT3a in TCL lines, demonstrating on-target activity of tolinapant and the HMAs, respectively. In addition, both azacytidine and decitabine increased the levels of RIPK3 up to 3-4 fold and 6-8 fold respectively, in a concentration dependent manner at 72Hrs by western blot analysis, indicating activation of the necroptosis pathway. Image:Figure 1: 3D synergy plots showing average synergy scores for decitabine in combination with tolinapant in the CTCL line HH.Summary/Conclusion: Tolinapant has demonstrated varying cytotoxic effects against a range of TCL lines as a single agent. Tolinapant, in combination with the HMAs, azacytidine and decitabine, displays a very high degree of synergism, in vitro, even at very low concentrations of both HMA drugs. The HMAs were able to activate the necroptosis pathway, as shown by the increase in RIPK3. Thus, activation of the necroptosis pathway, is a possible mechanism for the high degree of synergism displayed when HMAs are used in combination with tolinapant in the TCL lines in vitro. These data provide the rationale to initiate clinical trials with the combination of tolinapant and decitabine.

The combination of the IAP antagonist, tolinapant and hypomethylating agents (HMA) is highly synergistic in in vitro models of T-cell lymphoma (TCL).

e15088 Background: Peripheral T-cell lymphoma (PTCL) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas that often carry a poor prognosis with standard chemotherapy. Tolinapant is a potent antagonist of inhibitors of apoptosis proteins (cIAP1/2 and XIAP). In a phase 2 trial (NCT02503423), tolinapant showed activity against heavily pre-treated patients with TCL. We explored the sensitivity of a range of TCL lines to tolinapant, established the synergy coefficient and interrogated the mechanism of synergy between tolinapant and drugs active against PTCL including romidepsin, pralatrexate, and the HMAs; azacytidine and decitabine. Methods: A panel of 10 human TCL lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10 ng/ml of TNFα. For combination studies, each drug was tested, in the presence or absence of TNFα. Synergy coefficient was calculated using Excess over Bliss. Additionally, the on-target effects of the drugs were measured by analyzing levels of the IAPs, acetylated histones, DNMTs and key apoptosis and necroptosis markers by Western blotting. Results: TCL Lines demonstrated varying sensitivities to tolinapant with the most sensitive cell line, ALK+ ALCL SUP-M2, having an IC50 as low as 20 nM ± 1 nM while a resistant CTCL cell line HH had an IC50 of over 20 µM. In combination experiments using both cell lines, tolinapant plus azacytidine or decitabine displayed the highest degree of synergism, compared to romidepsin or pralatrexate. Of note, a high degree of synergism was also seen with concentrations 10 folds lower of azacytidine or 100 folds lower of decitabine, added daily (Table). The combination of tolinapant and the HMAs led to a decrease in the levels of cIAP1 and DNMT3a in TCL lines, demonstrating on-target activity of tolinapant and the HMAs, respectively. In addition, both azacytidine and decitabine increased the levels of RIPK3 and MLKL by western blot analysis, indicating activation of the necroptosis pathway. Conclusions: Tolinapant has demonstrated varying cytotoxic effects against a range of TCL lines as a monotherapy and displays a very high degree of synergism in combination with the HMAs, azacytidine and decitabine, also at low concentration, in vitro. The HMAs were also able to activate the necroptosis pathway, providing a possible mechanism for the high degree of synergism displayed when combined with tolinapant in the TCL lines in vitro. These data provide the rationale to initiate phase I trials with the combination of tolinapant and HMAs. Average synergy scores at 48 hrs for drugs used in combination with tolinapant in a CTCL line HH and ALK+ ALCL line SUP-M2. Above 10 indicates synergy.[Table: see text]

Combination of Resminostat with Ruxolitinib Exerts Antitumor Effects in the Chick Embryo Chorioallantoic Membrane Model for Cutaneous T Cell Lymphoma.

Simple SummaryThe combination of Resminostat (HDACi) and Ruxolitinib (JAKi) exerted cytotoxic effects and inhibited proliferation of CTCL cell lines (MyLa, SeAx) in vitro. The aim of the present study was to validate their antitumor effects in vivo using the chick embryo chorioallantoic membrane (CAM) model, which allows quick and efficient monitoring of tumor growth, migration, invasion, and metastatic potential. The drug combination exhibited a significant inhibition of primary tumor size, and inhibited intravasation and extravasation of tumor cells to the liver and lung. It also exerted an inhibitory effect in the migration and invasion of tumor cells and significantly reduced key signaling pathway activation. Our data demonstrate that the CAM assay could be employed as a preclinical in vivo model in CTCL for pharmacological testing, and that the combination of Resminostat and Ruxolitinib exerts significant antitumor effects in CTCL progression that need to be further evaluated in a clinical setting.The combination of Resminostat (HDACi) and Ruxolitinib (JAKi) exerted cytotoxic effects and inhibited proliferation of CTCL cell lines (MyLa, SeAx) in previously published work. A xenograft tumor formation was produced by implanting the MyLa or SeAx cells on top of the chick embryo chorioallantoic membrane (CAM). The CAM assay protocol was developed to monitor the metastatic properties of CTCL cells and the effects of Resminostat and/or Ruxolitinib in vivo. In the spontaneous CAM assays, Resminostat and Ruxolitinib treatment inhibited the cell proliferation (p < 0.001) of MyLa and SeAx, and induced cell apoptosis (p < 0.005, p < 0.001, respectively). Although monotherapies reduced the size of primary tumors in the metastasis CAM assay, the drug combination exhibited a significant inhibition of primary tumor size (p < 0.0001). Furthermore, the combined treatment inhibited the intravasation of MyLa (p < 0.005) and SeAx cells (p < 0.0001) in the organs, as well as their extravasation to the liver (p < 0.0001) and lung (p < 0.0001). The drug combination also exerted a stronger inhibitory effect in migration (p < 0.0001) rather in invasion (p < 0.005) of both MyLa and SeAx cells. It further reduced p-p38, p-ERK, p-AKT, and p-STAT in MyLa cells, while it decreased p-ERK and p-STAT in SeAx cells in CAM tumors. Our data demonstrated that the CAM assay could be employed as a preclinical in vivo model in CTCL for pharmacological testing. In agreement with previous in vitro data, the combination of Resminostat and Ruxolitinib was shown to exert antitumor effects in CTCL in vivo.

Targeting CD70 in cutaneous T-cell lymphoma using an antibody-drug conjugate in patient-derived xenograft models

AbstractCD70 is a member of the tumor necrosis factor receptor superfamily. Emerging data indicate that CD70 may be a suitable target for various malignancies. We investigated the expression of CD70 in cutaneous and systemic T-cell lymphomas and conducted preclinical studies of SGN-CD70A, a CD70-directed antibody-drug conjugate (ADC), using patient-derived xenograft cutaneous T-cell lymphoma (CTCL PDX) models. CD70 expression was examined by immunohistochemical (IHC) stains in 49 diagnostic specimens of T-cell lymphomas. The activities of SGN-CD70A in growth inhibition and apoptosis induction were examined in CTCL cell lines and primary CTCL tumor cells. Using previously established CTCL PDXs, we conducted a dose-finding trial followed by a phase 2-like trial to evaluate the optimal dosing and the efficacy of SGN-CD70A in tumor-bearing PDX animals. The therapeutic efficacy of SGN-CD70A was measured by tumor-associated cell-free DNA (cfDNA) and survival of treated PDXs. We found that CD70 is highly expressed in T-cell lymphomas, especially in CTCL. SGN-CD70A inhibited cell growth and induced apoptosis in CD70-expressing CTCL cell lines and primary tumors cells. Additionally, SGN-CD70A at 100 μg/kg and 300 μg/kg prolonged the survival of PDXs in a dose-dependent manner. Finally, treatment with 3 doses of SGN-CD70A at 300 μg/kg was superior to a single-dose treatment in survival prolongation (median survival: 111 days vs 39 days; P = .017). Most importantly, multiple dosing of SGN-CD70A induced complete eradication of established tumors in PDXs measured by cfDNA. Our results demonstrated marked antitumor activity of SGN-CD70A in CTCL PDXs, providing compelling support for its clinical investigation.

Chimerized Anti-ICOS 314.8 Monoclonal Antibodies Inhibit Tumor Cells and Regulatory T Cells in Patients with Sézary Syndrome

Background: In a previous study, we reported the strong expression of Inducible T-cell costimulatory (ICOS) by Sézary cells, and presented the excellent preclinical efficacy results of anti-ICOS antibody drug conjugates (ADCs) in both Sézary syndrome (SS) and angioimmunoblastic T-cell lymphoma. Although exerting a potent direct action on the tumor cell, ADCs have the disadvantage of being associated with a cumulative toxicity, related to the conjugated drug. The development of antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP)-inducing anti-ICOS monoclonal antibodies (mAbs) is therefore of great importance to ensure long-term maintenance therapy.Methods: We first determined which anti-ICOS clone was the best candidate to induce an ADCC effect on ICOS + cell lines (MyLa, MJ and HUT78), using Mouse FcγRIII ADCC Bioassay (Promega®). The selected mAb was then chimerized and afucosylated (GlymaxX® technology, Evitria®).Secondly, we evaluated in vitro ADCC and ADCP effect of the chimeric anti-ICOS mAb against ICOS + CTCL cell lines, compared to both positive controls (mogamulizumab (moga) and alemtuzumab) and negative controls (IgG1 isotype control, and rituximab). To perform ADCC experiments, we co-incubated target cells with mAbs and allogenic NK lymphocytes from healthy volunteers (HV). Cellular apoptosis was measured by flow cytometry using the Caspase 3/7 assay (Promega®). For ADCP, monocytes were sorted from HV blood samples and treated with M-CSF. Target cells were labeled with PKH67 (Sigma-Aldrich®) and after co-incubation, CD14 +CD11b +PKH67 + monocytes were analyzed by flow cytometry.Finally, we verified the ADCC/ADCP potency of anti-ICOS mAbs on primary Sézary cells isolated from 16 moga-naïve SS patients, and 6 patients who had developed resistance to moga. We also questioned whether anti-ICOS mAbs were able to promote the autologous apoptosis of Sézary cells and T regulatory cells (Tregs) when directly incubated with peripheral blood mononuclear cells (PBMCs) from patients with SS.Results: Among 7 different anti-ICOS clones, 314.8, 92.17 and 293.1 clones had the higher ADCC activity against MyLa, MJ and HUT78. Of these 3 clones, 314.8 had the best affinity to the receptor, and the best ability to inhibit binding between ICOS receptor and a recombinant ICOS ligand protein. Anti-ICOS 314.8 mAb was then chosen to be chimerized and glyco-engineered.ICOS and CCR4 were strong and similarly expressed on MyLa and MJ. HUT78 had only mild expression of ICOS and CD52. Anti-ICOS mediated potent ADCC of cell lines (respectively 39.1±5% and 52.6±2.4% for MyLa and MJ cells), without significant difference when compared to mogamulizumab. In HUT78 cells, anti-ICOS induced a specific apoptosis of 35.7±5% versus 15.6±5.6% with alemtuzumab (p=0.02; CI95%: 4.1-36.1).Moreover, phagocytosis induced by anti-ICOS was significantly increased than that induced by negative controls. On MyLa cells, anti-ICOS had a greater phagocytosis activity than moga (59.4±5.2% vs 39.4±5.1%, p=0.031).Expression of ICOS by circulating tumor cells was found in all the 16 moga-naïve patients. The expression was strong, as 61±6% of tumor cells expressed ICOS vs 20±8% of non-tumoral CD4 + cells. CCR4 was more expressed than ICOS on both Sézary cells and non-tumoral CD4 + cells (91±6%, and 44±9% respectively). Anti-ICOS induced the apoptosis of 57.1±4.7% Sézary cells, compared to 16.9±2.2% with rituximab (p<0.01; CI95%:-53–27). The efficacy was better than with alemtuzumab, but there was no significant difference with moga. The ADCP effect induced by anti-ICOS did not differ with moga. Interestingly, anti-ICOS were effective in 6 moga-resistant patient, as they induced 38.9±5.9% of apoptosis, compared to 12.4±4.7% with moga (p<0.001).Ex vivo, anti-ICOS allowed 39.4±19.9% and 70.1±20.1% lysis of Sézary cells and Tregs respectively, with no difference with moga and alemtuzumab. However, the depletion of non-tumoral CD4 + and total PBMCs was significantly lower with anti-ICOS mAbs than with moga and alemtuzumab.Discussion: In a recent study, we showed that Treg cells of patients with SS have a high expression of ICOS. Here, we demonstrate that anti-ICOS mAbs induce Tregs depletion, which may improve immune profiles and emphasize Sézary cells killing. Our data suggest robust anti-tumor activity of anti-ICOS mAbs in SS, and xenograft experiments are underway to confirm these findings. DisclosuresLopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Tolinapant (ASTX660), a Non-Peptidomimetic Antagonist of cIAP1/2 and XIAP, and the HDAC Inhibitor Romidepsin Are Synergistic in in Vitro Models of T Cell Lymphoma

Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). [Display omitted] DisclosuresSmyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

Abstract 1844: A CD70 antibody-drug conjugate is highly active and induces long term remission in patient-derived xenograft mouse models of cutaneous T cell lymphoma

Abstract Cutaneous T cell lymphoma (CTCL) is a type of non-Hodgkin's lymphoma in which malignant T cells initially home to the skin followed by systemic dissemination as the disease progresses. CD70 is rarely expressed in normal tissues but is overexpressed in activated T and B lymphocytes. From archival diagnostic specimens of 37 patients, we demonstrate that CD70 is highly expressed in subtypes of mature TCL, especially CTCL. SGNCD70A is a new anti-CD70 antibody-drug conjugate combining a monoclonal antibody to CD70 with a DNA-damaging agent. SGNCD70A inhibited cell growth and induced apoptosis of CD70+ CTCL cell lines and primary tumors from patient-derived xenograft (PDX) mice, but had no inhibitory effect on CD70- lymphoblastic TCL cells and normal T cells. Next, we examined the anti-tumor activity of SGNCD70A in CTCL PDX model. One week after tumor inoculation, mice were treated intraperitoneally with single-dosing of PBS, 100 μg/kg (SGN70A100) or 300 μg/kg (SGN70A300) of SGN-CD70A. SGN-CD70A prolonged survival of treated mice in a dose-dependent fashion compared to untreated mice. Median survival was 21 days, 27.5 days (P=0.041), and 57 days (p&amp;lt;0.01) for the PBS control, SGN70A100 and SGN70A300 group, respectively. To confirm that survival was dictated by disease progression, rather than drug toxicity of SGN-CD70A, we measured tumor burden by cell-free DNA (cfDNA) throughout the course of treatment. We demonstrated that cfDNA paralleled the disease progression and peaked before death of the animals. In contrast, in surviving mice, cfDNA remained at the background level. Importantly, we discovered relapse occurred in SGN70A300 group (3 of 4, 75%) due to persistent disease, not due to acquired resistance to SGN-CD70A. Thus, we investigated whether multiple dosing could lead to complete regression of tumors in PDX model. We treated PDX mice with SGN70A300 weekly (SGN70A300x3) for 3 consecutive weeks. In the SGN70A300x3 group, three of four mice survived beyond day 90 (median &amp;gt;85 days), resulting in markedly prolonged survival compared to the single-dose- and PBS-treated mice with a median survival of 39 days, (P&amp;lt;0.01) and 20 days (P&amp;lt;0.01), respectively. Compared to PBS group, both SGN70A300 and SGN70A300x3 treated mice had delayed tumor growth and cfDNA rise. Furthermore, in SGN70A300 group, cfDNA started to rise seven days before the endpoint of the mice in a range of 30-42 days, while three surviving mice in SGN70A300x3 remained at background cfDNA levels until day 85, indicating that multiple doses of SGN-CD70A decreased overall tumor burden and the occurrence of relapse in CTCL. Our results showed that CD70 is highly expressed in mature TCL, especially in CTCL. Using CTCL PDX models, SGN-CD70A has marked anti-tumor activity, leading to long-term survival of treated mice. Our results provide a rationale for clinical investigation of SGN-CD70A in patients with TCL. Citation Format: Chi-Heng Wu, Chen-Yen Yang, Linlin Wang, Ryan Gill, Frank McCormick, Weiyun Z. Ai. A CD70 antibody-drug conjugate is highly active and induces long term remission in patient-derived xenograft mouse models of cutaneous T cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1844.

Edo-S101, a Bendamustine (BDM)/ Histone-Deacetylase Inhibitor (HDACi) Fusion Molecule, Demonstrates Potent Preclinical Activity Against T-Cell Malignancies and Overcomes BDM-Resistance

Introduction: T-cell and natural killer (NK)-cell lymphomas encompass a heterogeneous spectrum of malignancies with variable degrees of biological and clinical aggressiveness and different patterns of nodal/extra nodal dissemination. Despite advances in understanding of their genetic landscape, treatment options for these tumors are limited and survival remains dismal especially in patients (pts.) who relapse/progress after frontline treatment. Therefore, development of new active agents is a pressing medical need in this setting. The unconventional alkylator BDM and HDACi are clinically effective in pts. with recurring disease and the combination of these agents was suggested as a way to overcome early resistance and improve response and survival rates in T-cell tumors. Since EDO-S101 is a fusion molecule that combines the active structures of BDM and the pan-HDACi vorinostat to simultaneously damage DNA and block damage repair, we investigated its antitumor activity in preclinical models of human T- and NK- lymphomas.Methods: We assessed the patterns of EDO-S101 cytotoxicity and its regulatory effects on genes involved in DNA-damage/repair, apoptosis and drug resistance on a large panel of cell lines representative of most T-lymphoma subtypes, including precursor T-cell lymphoma/leukemia (pTCL; MOLT-4, KE-37, Jurkat, CCRF-CEM), anaplastic large T-cell lymphoma (ALCL; SR-786, SUDHL1) and cutaneous T-cell lymphoma (CTCL; HUT78, MYLA, HH, PB2B, MAC-2A). As a further model, we exploited an ALCL cell line (SUDHL1-R100) selected for resistance to BDM. SUDHL1-R100 display a growth pattern indistinguishable from parental cells in the presence of BDM (100 μmol/L).Results: EDO-S101 induced a significant time- and dose-dependent inhibition of growth and survival in all T-cell lines, with a IC50 at 48 hrs (0.33 to 0.68 μmol/L), that was at least 90% and 50% lower than IC50 of BDM (10.5 to 23.67 μmol/L) and vorinostat (0.42 to 1.17 μmol/L) in the same cell lines. No significant differences emerged across various cell lines as to sensitivity to EDO-S101. Notably, growth inhibitory effects of EDO-S101 were 35% to 40% higher than those achieved by the concomitant exposure of tumor cells to BDM and vorinostat at equimolar concentrations. A significant induction of the DNA double strand breaks marker pγH2AX, along with decreased levels of Thr-1989 pATR and Polo-like kinase 1 (PLK1) were consistently observed in pTCL, ALCL and CTCL cell lines, indicating that EDO-S101 triggered inappropriate mitotic entry with unrepaired DNA damage. In addition, qRT-PCR results showed upregulation of the proapototic genes NOXA, p21 and/or p27 in pTCL, CTCL and ALCL cells. Among DNA repair genes, EDO-S101-induced downregulation of ATM, ATR and EXO1 was paralleled by an increase of MGMT transcripts. Most intriguingly, BDM-resistant ALCL cells (SUDHL1-R100) were highly sensitive to EDO-S101, with an IC50 of only 0.72 μmol/L, but less responsive to vorinostat (IC50: 2.17 μmol/L) than parental cells (IC50: 1.46 μmol/L). Exposure to EDO-S101 also resulted in transcriptional regulation of components of the PI3K pathway, mainly the PI3Kα and δ isoforms and mTOR. These findings, prompted experiments showing that EDO-S101 at sublethal concentrations was highly synergic with the predominant PI3Kα/δ inhibitor copanlisib in inducing cell death of tumor T- and NK-cells. Finally, exposure to EDO-S101 (1.0 μmol/L) for 48 hrs resulted in a sustained upregulation of surface PD1 and PD-L1 in most tumor T- and NK-cell lines, as a possible result of its HDACi component.Conclusions: We have shown for the first time that the BDM/vorinostat fusion molecule EDO-S101 is very active in preclinical models of T/NK cell lymphomas. It shows a superior activity towards different types of tumor T- and NK-cells than its single components administered either individually or in combination by also overcoming acquired resistance to BDM. Data indicate that EDO-S101 simultaneously damages DNA and interferes on its repair pathways in tumor T-cells. We also showed synergy between EDO-S101 and PI3Kα/δ inhibitors possibly due to regulatory effects on specific components of the PI3K pathway as well as a potential for this agent to enhance PD1-blockade strategies. A clinical trial is ongoing to evaluate efficacy and safety of EDO-S101 in pts. with relapsed/refractory hemopoietic malignancies including T-cell lymphomas. DisclosuresMehrling:Mundipharma-EDO Gmbh, Basel, CH: Employment. Pinto:Celgene: Honoraria; Merck Sharp Dome: Honoraria; Millenium Takeda: Research Funding; Roche: Honoraria; Bristol Myers Squibb: Honoraria; Gilead: Honoraria; Helssin: Honoraria; Mundipharma EDO: Speakers Bureau.

Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma.

The combination of JAK/STAT and HDAC inhibitors exerted beneficial effects in haematological malignancies, presenting promising therapeutic CTCL targets. We aim to investigate the efficacy of JAK1/2i ruxolitinib in combination with HDACi resminostat in CTCL in vitro. Non-toxic concentrations of ruxolitinib and/or resminostat were administered to MyLa (MF) and SeAx (SS) cells for 24h. Cytotoxicity, cell proliferation and apoptosis were estimated through MTT, BrdU/7AAD and Annexin V/PI assay. Multi-pathway analysis was performed to investigate the effect of JAK1/2i and/or HDACi on JAK/STAT, Akt/mTOR and MAPK signalling pathways. Both drugs and their combination were cytotoxic in MyLa (p<0.05) and in SeAx cell line (p<0.001), inhibited proliferation of MyLa (p<0.001) and SeAx (p<0.001) at 24h, compared to untreated cells. Moreover, combined drug treatment induced apoptosis after 24h (p<0.001) in MyLa, and SeAx (p<0.001). The combination of drugs had a strong synergistic effect with a CI<1. Importantly, the drugs' combination inhibited phosphorylation of STAT3 (p<0.001), Akt (p<0.05), ERK1/2 (p<0.001) and JNK (p<0.001) in MyLa, while it reduced activation of Akt (p<0.05) and JNK (p<0.001) in SeAx. The JAKi/HDACi combination exhibited substantial anti-tumor effects in CTCL cell lines, and may represent a promising novel therapeutic modality for CTCL patients.

JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL

AbstractCutaneous T-cell lymphoma (CTCL) is a malignancy of skin-homing T lymphocytes that is more likely to involve the peripheral blood in advanced stages. For such patients with advanced disease, there are few available systemic treatment options, and prognosis remains poor. Exome sequencing studies of CTCL have suggested therapeutic targets, including within the JAK/STAT pathway, but JAK inhibition strategies may be limited by patient-specific mutational status. Because our recent research has highlighted the potential roles of single and combination approaches specifically using BCL2, bromodomain and extra-terminal domain (BET), and histone deacetylase (HDAC) inhibition, we aimed to investigate the effects of JAK inhibition on CTCL cells and established CTCL cell lines when paired with these and other targeting agents. Peripheral blood malignant CTCL isolates exhibited differential responses to JAK inhibition, with JAK2 expression levels negatively correlating to 50% inhibitory concentration (IC50) values. Regardless of single-agent sensitivity, JAK inhibition potentiated malignant cell cytotoxicity in combination with BCL2, BET, HDAC, or proteasome inhibition. Combination inhibition of JAK and BCL2 showed the strongest potentiation of CTCL cytotoxicity, driven by both intrinsic and extrinsic apoptosis pathways. JAK inhibition decreased expression of BCL2 in the high-responder samples, suggesting a putative mechanism for this combination activity. These results indicate that JAK inhibition may have major effects on CTCL cells, and that combination strategies using JAK inhibition may allow for more generalized cytotoxic effects against the malignant cells from patients with CTCL. Such preclinical assessments help inform prioritization for combination targeted drug approaches for clinical utilization in the treatment of CTCL.