Subgroups of T-Cell Prolymphocytic Leukemia (T-PLL) Discovered By High-Throughput Ex Vivo Drug Testing and Genetic Profiling

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare disease with an aggressive clinical course and a median overall survival of less than three years. Although almost 75% of T-PLL patients are reported to harbor translocations causing the activation of the proto-oncogene TCL1A, T-PLL is genetically heterogenous: most T-PLL patients also have mutations or deletions in the ATM gene and the genes involved in the JAK-STAT pathway are mutated in 76% of cases. There is an urgent need for more rational based therapies, but clinical trials are difficult to perform due to the rareness of the disease. Here, we systematically explored the diversity of drug responses in T-PLL patient samples ex vivo using a drug sensitivity and resistance testing (DSRT) system including 306 oncology drugs (approved or investigational). We also aimed to determine any associations between the genetic aberrations and drug sensitivities in T-PLL patients. Primary mononuclear cells were gathered from 30 T-PLL patients for drug testing. Cells were plated in 384-well plates and subjected to the 306 substances using a 10,000-fold concentration range. After 72 hours, cell viabilities were measured, the results were depicted as dose-response curves for each compound, and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were computed by comparing patient samples to healthy donors. Drug response profiles across patients were clustered and visualized by hierarchical clustering. The subgroups resulting from the clustering were statistically compared using a two-sample t-test to understand which drug classes were driving the grouping. To delineate heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by targeted amplicon sequencing of tumor cells to profile known recurrent genetic variants (STAT5b, IL2RG, JAK1, JAK3, ATM). Information on chromosomal aberrations (TCL1A translocations, ATM deletions) was derived from parallel clinicopathologic databases. Amplicon sequencing revealed that 70% of T-PLL patients (21/30) harbored a mutation in genes involved in the JAK-STAT pathway (JAK1, JAK3, STAT5b or IL2RG). The most prevalent mutation led to an M511I amino acid exchange in the JAK3 protein (26% of patients). Interestingly, the STAT5b mutations (5/30) did not coexist with any of the JAK mutations in our cohort. Based on DSRT analysis, all T-PLL samples were sensitive to the CDK-inhibitor SNS-032 and the anti-cancer antibiotic actinomycin D. Next, we clustered patients using sDSS values for all 306 different substances, and this showed that patient samples could be divided into 3 main groups, based on their drug responses (Figure). According to two-sample t-test, the grouping was driven by the selective sensitivities of Groups II and III to HDAC inhibitors (belinostat, panobinostat, quisinostat, CUDC-101 and vorinostat) and the selective sensitivity of Group III to PI3K/AKT/mTOR inhibitors (AZD-8055, MK-2206, apitolisib, dactolisib, PF-04691502, ZSTK474, and omipalisib), HSP90 inhibitors (BIIB021, luminespib, alvespimycin, and tanespimycin) as well as JAK inhibitors (ruxolitinib, momelotinib, tofacitinib, gandotinib). Group I samples were on the other hand relatively resistant to these classes of drugs. Surprisingly, despite the prevalence of the signature event of activation of TCL1 (the established AKT coactivator) in nearly all cases, only a subset of cases (group III) responded to PI3K/AKT/mTOR inhibitors. Strikingly, the grouping of selective responses to HDAC, JAK, PI3K/mTOR/Akt and HSP90 inhibitors did not link to the presence of JAK/STAT mutations, TCL1A translocations, or ATM deletion status. Ex vivo drug screening of primary T-PLL samples revealed heterogenous selective drug responses in specific drug classes (such as HDAC-, JAK-, HSP90- and PI3K/Akt/mTOR-inhibitors). Surprisingly, the drug response patterns did not correlate with known recurrent genetic aberrations suggesting that sequencing for recurrent genetic biomarkers cannot easily be turned into effective therapeutic strategies in T-PLL, and that further elucidation of the biological pathways driving T-PLL is needed. Figure 1. Mutation status and clustering of HDAC-, PI3K/mTOR/Akt-, HSP90-, and JAK-inhibitor responses in PLL patients based on sDSS values Figure 1. Mutation status and clustering of HDAC-, PI3K/mTOR/Akt-, HSP90-, and JAK-inhibitor responses in PLL patients based on sDSS values Disclosures Koschmieder: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding; Novartis Foundation: Research Funding; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Other: Travel reimbursement for scientific conferences ; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences. Wennerberg:Pfizer: Honoraria, Research Funding. Ding:Merek: Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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Stroma-Derived Factors Significantly Impact the Drug Response Profiles of Patient-Derived Primary AML Cells: Implications for Drug Sensitivity Testing

Blood ◽

10.1182/blood.v124.21.3505.3505 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3505-3505

Author(s):

Riikka Karjalainen ◽

Tea Pemovska ◽

Muntasir Mamun Majumder ◽

David Tamborero ◽

Bhagwan Yadav ◽

...

Keyword(s):

Board Of Directors ◽

Conditioned Medium ◽

Research Funding ◽

Drug Testing ◽

Stromal Cell ◽

Kinase Inhibitors ◽

Ex Vivo ◽

Tumor Stroma ◽

Advisory Committees ◽

Equity Ownership

Abstract Background: Bone marrow (BM) microenvironment plays an important role in development of drug resistance in acute myeloid leukemia (AML) by supporting survival of therapy resistant leukemic cells that eventually may lead to disease relapse. Consideration of tumor stroma factors is therefore critically important when assessing the efficacy of drugs in the ex vivo drug testing of primary AML cells. To study the effects of tumor stroma factors on the response of AML patient cells ex vivo to a panel of cancer drugs, we evaluated the effects of stroma-derived conditioned medium (CM) on the sensitivity of genomically defined primary AML cells. Methods: Primary AML cells were isolated by Ficoll gradient separation from BM aspirates or peripheral blood of AML patients (n=13). For drug testing, 303 small molecule inhibitors were plated on 384-well plates in 5 different concentrations over a 10,000-fold concentration range. The stromal-cell conditioned medium (CM) was made by culturing human bone marrow (BM) stromal cell line HS-5 (American Type Culture Collection) in RPMI 1640 medium for 3 days. AML cells were added to the plates in either CM diluted with RPMI 1640 medium (25% CM) or in mononuclear cell medium (MCM, Promocell), which was used as the standard medium comparison. Cell viability was measured after 72 h and dose response curves generated for each drug. Drug sensitivity scores (DSS) were calculated as described previously (Yadav et al, 2014). Phosphorylation profiles of 43 proteins were analyzed with a human phospho-kinase array (R&D Systems). In addition, somatic mutations were identified by exome sequencing using DNA from the leukemia cells and matched skin biopsies, while expressed fusion genes were identified by transcriptome sequencing. Results: AML samples with activating mutations to kinases such as FLT3 or PDGFRB exhibited more sensitive ex vivo drug response profiles, particularly to broad-spectrum kinase inhibitors, compared to samples driven by other types of mutations. When the same AML samples were compared between the two conditions, CM or MCM, the drug sensitivities were different for many classes of drugs (Table 1). In CM, samples typically lost sensitivity to many of the tested drugs, such as topoisomerase II inhibitors, BCL2 inhibitors and several other classes of tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity in CM was particularly striking in the FLT3 and PDGFRB mutated cases. Cluster analysis of overall drug responses for AML samples tested in MCM resulted in a tight group of most TKIs, reflecting their overlapping target profiles. However, when the analysis was applied to responses from the same cells tested in CM, the TKI grouping was more dispersed. Thus, these results indicate that tyrosine kinase signaling is stringently regulated in standard medium, whereas CM helps to support cell survival resulting in lower responses to a range of TKIs. To test this hypothesis, phosphorylation of 43 different kinases was measured with AML samples incubated in either CM or MCM. CM induced phosphorylation of multiple proteins including p38α, HSP27, Src, Lyn, Hck and STAT6 proving the activation of other signaling pathways. Conclusions: Our dataindicate that stromal cell conditioned medium may have prominent effects on ex vivo drug responses of AML cells. BM factors likely provide survival cues that make primary patient-derived AML cells resistant to several targeted agents, such as topoisomerases and TKIs. This underscores the need to develop drug testing methods that take into account tumor-microenvironment interactions. Disclosures Gjertsen: BerGenBio AS: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim : Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wennerberg:Pfizer: Research Funding. Heckman:Celgene: Research Funding.

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Characterizing the Anti-Apoptotic Dependencies of T-Cell Prolymphocytic Leukemia Identifies HDAC and JAK/STAT Pathway Inhibitors As Promising Combination Partners to Augment Bcl-2 Targeted Killing By Venetoclax

Blood ◽

10.1182/blood-2019-126773 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 807-807 ◽

Cited By ~ 1

Author(s):

Charles Herbaux ◽

Christoph Kornauth ◽

Stéphanie Poulain ◽

Olivier Tournilhac ◽

Mary C Collins ◽

...

Keyword(s):

Cell Death ◽

Lymph Node ◽

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

Advisory Committees ◽

Prolymphocytic Leukemia ◽

Drug Treatments ◽

A Cell ◽

Stat Pathway

Introduction: Response to conventional therapies for patients with T-cell prolymphocytic leukemia (T-PLL) is usually poor and is associated with short survival. The BCL-2 antagonist venetoclax was recently found to have some clinical activity in this disease (B Boidol et al., Blood, 2017); however, these early data suggest that this drug will not provide prolonged response when given as monotherapy. Several other drug classes have demonstrated preclinical activity in T-PLL, including HDAC inhibitors (HDACi), JAK/STAT inhibitors (JAK/STATi), and TCR pathway inhibitors (TCRi), particularly ITK inhibitors. To determine which drug(s) may be the optimal combination partner(s) for venetoclax in T-PLL, we utilized a functional approach known as BH3 profiling. This assay measures how close a cell is to the threshold of apoptosis ("priming") and identifies which anti-apoptotic proteins a cell depends on for survival. We also utilized a variant known as dynamic BH3 profiling (DBP) to measure early changes in pro-apoptotic signaling after various drug treatments. Methods: Clinically annotated primary T-PLL patient samples were obtained from the French Innovative Leukemia Organization network after informed consent. Peripheral blood mononuclear cells were isolated by Ficoll and viably frozen and later thawed for the experiments. Baseline BH3 profiling to measure cytochrome C (cyto-C) release was performed as per Ryan et al., Methods, 2013, and DBP as per Montero et al., Cell, 2015. Viability was assessed by AnnexinV/Hoechst staining. Ex vivo drug treatments included: BH3 mimetics (BCL-2i: venetoclax (VEN), MCL-1i: AZD5991, S63845), HDACi (belinostat = BEL), JAK/STATi (ruxolitinib = RUX) and TCRi (PRN694 = PRN). Protein expression was assessed by standard Western Blot. Primary CLL cells were used in some experiments as a comparator. To mimic the lymph node microenvironment, DBP and viability assays were performed in co-culture with the stromal cell line NK.tert. Tumoral DNA was also extracted, and we performed NGS on a panel of 29 genes, including ATM and TP53, as well as Sanger sequencing to assess for IL2R, JAK1, JAK3, STAT5B mutations. Statistical analyses were by unpaired and paired t-test with a two-tailed nominal p ≤ 0.05 considered as significant. Results: Samples were evaluated from 31 T-PLL patients. Baseline BH3 profiling revealed that, compared to CLL cells, T-PLL cells are less primed for apoptosis but have comparable dependency on MCL-1. BCL-2 dependency was found to be significantly lower in T-PLL than CLL (cyto-C release 48.8%; 62.7% p=0.0005), and to decrease further in the presence of stroma (Figure A, cyto-C release from 72.6% to 36.2%, p = 0.01). Consistent with our BH3 profiling results, the degree of BCL-2 dependency in T-PLL cells was strongly associated with the amount of apoptotic cell death induced by VEN (R2 -0.58, p=0.004), whereas MCL1 dependency was strongly associated with the cell death induced by the MCL1 inhibitors S63845 and AZD5991 (R2 -0.59, p=0.002 and R2 -0.68, p=0.0005 respectively, Figure B). We next performed DBP to assess the changes in apoptotic priming in T-PLL cells induced by HDACi, JAK/STATi and TCRi. To utilize doses similar to what can be achieved in patients, we assessed BEL 1mM, RUX 1mM and PRN 1mM. BEL and RUX increased overall T-PLL cell priming and BCL2 dependency (delta cyto-C release of 26.8%, p=0.004 and 14.8%, p=0.01 respectively Figure C), with no effect on MCL1 dependency. PRN had no significant effect on priming. Consistent with the DBP data, our viability assays showed that BEL and RUX induced significantly more cell death when combined with VEN compared to PRN (Figure D). Mutations in ATM, TP53, and JAK/STAT pathway genes were observed in cells from 35%, 6%, and 53% of patients, respectively, and did not impact the ex vivo activity of these drugs. Conclusion: We report the first data for BH3 profiling in T-PLL. We found that this disease is heterogeneously dependent on both BCL-2 and MCL-1, and that the lymph node microenvironment may decrease BCL-2 dependency. HDACi and JAK/STATi both enhance BCL-2 dependence, thereby sensitizing T-PLL cells to VEN. Ongoing studies will help further define the mechanism underlying these promising new combinations for T-PLL. Disclosures Herbaux: BMS: Honoraria; Gilead: Honoraria; Takeda: Honoraria; Abbvie: Honoraria; Janssen: Honoraria. Valentin:Roche: Other: Travel reimbursement; Abbvie Inc: Other: Travel reimbursement. Morschhauser:F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Bayer: Membership on an entity's Board of Directors or advisory committees; Epizyme: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Staber:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda-Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; MSD: Honoraria, Speakers Bureau. Davids:AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding; AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria.

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High-Throughput Functional Ex-Vivo Drug Testing and Multi-Omics Profiling in Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-124332 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4641-4641

Author(s):

Tom Erkers ◽

Brinton Seashore-Ludlow ◽

Nona Struyf ◽

Francesco Marabita ◽

Tojo James ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Drug Testing ◽

Ex Vivo ◽

University Hospital ◽

Advisory Committees ◽

Drug Responses ◽

Acute Myeloid

Acute myeloid leukemia (AML) is the most common leukemia in adults and the prognosis is dismal. The heterogeneity of driver mutations in AML from one patient to another is significant. Although new treatment targets and modalities have recently been introduced, a lack of therapies to cover the entire spectrum of molecularly distinct AML classes remains a challenge. Furthermore, often only a fraction of the patients carrying mutations actually respond to the drugs predicted to target such genomic subtypes in AML. Our question was if outcome can be improved by optimizing existing and emerging therapies? To address this, we applied direct functional drug testing ex vivo in patients with newly diagnosed AML, along with deep molecular profiling to identify new therapeutic and diagnostic opportunities for specific genetic subclasses. To date, 122 patient samples have been included in the study. We have collected AML patient mononuclear cells isolated from fresh bone marrow aspirates from the Karolinska University Hospital Huddinge and Uppsala University Hospital, or applied biobanked samples from the Swedish acute leukemia biobank. The cells from the patient are tested for 72h with up to 525 conventional and investigative oncology drugs in a 5-point concentration range to determine optimal treatment options. The test is based on bulk cell viability and the cells are cultured in bone marrow stroma-conditioned media. The data is compared to healthy bone marrow controls to filter out generally toxic drugs and combinations. Excess patient material is applied for multi-omics analysis, including genome sequencing, deep proteomic profiling, concentrations of soluble factors, bulk/single-cell RNA sequencing and mass cytometry. Our results indicate significant heterogeneity in functional drug responses across individual patients, even among those with the same founder mutations. We could identify potential targeted treatments for most patients based on the exvivo testing. For instance, patients that show resistance to the Bcl-2 inhibitor Venetoclax can be associated with a distinct drug sensitivity response pattern that includes drugs with different mechanisms of actions. Based on the clustering of drug response data in all clinical samples, we could identify groups of patients with similar global drug responses, and drugs with similar patterns of efficacy across all patients. Disclosures Lehmann: Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbive: Membership on an entity's Board of Directors or advisory committees; TEVA: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kallioniemi:Medisapiens: Other: Co-founder and stockholder; Sartar Therapeutics: Other: Co-founder and stockholder; Astra-Zeneca: Other: Joint Grant; Pelago: Other: Joint Grant; Takara: Other: Joint Grant; Abbot: Other: Licensing income.

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DEPTOR Is a Regulator of Response to mTOR Kinase Inhibitors in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.2916.2916 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2916-2916

Author(s):

Diana Cirstea ◽

Teru Hideshima ◽

Loredana Santo ◽

Homare Eda ◽

Miriam Canavese ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Kinase Inhibitors ◽

Mtor Inhibitors ◽

Mtor Pathway ◽

Differential Sensitivity ◽

Advisory Committees ◽

Mtor Kinase ◽

Regulatory Cascade

Abstract Abstract 2916 Inhibition of the PI3K/mTOR pathway is a promising therapeutic strategy in targeting multiple myeloma (MM) cells in the bone marrow (BM) microenvironment, which abnormally activates PI3K/mTOR signaling cascade mediating proliferation, anti-apoptosis and drug resistance. Exploring the targeting of PI3K/mTOR pathway has led to the development of different therapeutic approaches; however, mTORC1 inhibitors (i.e., temsirolimus and everolimus) have demonstrated only modest activity as single agents. In this regard, several mechanisms underlying rapamycin resistance, including mTOR/S6K1-mediated feedback loops resulting in activation of PI3K/Akt and ERK signaling, have been proposed. Importantly, recent studies have identified mTOR kinase and the mTOR-DEPTOR counter-regulatory cascade as key mediators of mTORC1 and mTORC2 multi-protein complexes, with differential sensitivity to rapamycin. Indeed, targeting DEPTOR/mTORC1/mTORC2 signaling by inhibition of mTOR kinase proved an effective strategy to overcome some of the limitations of TORC1 inhibition in MM cells, evidenced in our studies of the novel dual mTORC1 and mTORC2 selective inhibitor AZD8055. Unlike rapamycin, AZD8055 induced apoptosis and inhibited MM cell growth even when co-cultured with cytokines (i.e., IL-6, IGF1) or BMSCs, presumably through simultaneous suppression of mTORC1 and mTORC2 signaling including the rapamycin-resistant 4E-BP1 (downstream of mTORC1) and Akt as well as NDRG1 (effectors of mTORC2). We examined mRNA and protein level of DEPTOR in MM cell lines treated with AZD8055 versus rapamycin and observed no significant changes. To examine the functional significance of DEPTOR in response to mTOR inhibitors, we utilized lentiviral shRNA to knockdown DEPTOR in OPM1 MM cells. DEPTOR-knockdown cells acquired resistance to AZD8055 treatment, suggesting that DEPTOR is a key modulator of mTORC1/2 signaling. Moreover, DEPTOR knockdown triggered decrease in Akt phosphorylation (Ser473), associated with suppression of Rictor phosphorylation (Thr1135). DEPTOR co-immunoprecipitation with Rictor was also abrogated by both AZD8055 and rapamycin treatment. Taken together, our results indicate the role of DEPTOR, either alone or as an mTOR/Rictor interacting molecule, in mediating the anti-MM activity induced by mTOR kinase inhibitors in MM cells. These data therefore both provide insights into the molecular profiles that may predict sensitivity/resistance to second generation of mTOR inhibitors in MM, and may be useful to select MM patients for mTOR inhibitor therapy. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Guichard:AstraZeneca, UK: Employment, Shares from AstraZeneca, UK. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

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Dose Dependent Enhancement Of Neutrophil Recovery By Infusion Of Notch Ligand Ex Vivo Expanded Cord Blood Progenitors: Results Of a Multi-Center Phase I Trial

Blood ◽

10.1182/blood.v122.21.297.297 ◽

2013 ◽

Vol 122 (21) ◽

pp. 297-297 ◽

Cited By ~ 4

Author(s):

Colleen Delaney ◽

Filippo Milano ◽

Ian Nicoud ◽

Shelly Heimfeld ◽

Chatchada Karanes ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Graft Failure ◽

Ex Vivo ◽

Chronic Gvhd ◽

Advisory Committees ◽

Neutrophil Recovery ◽

Cd34 Cells ◽

Primary Graft Failure

Abstract Introduction There is a strong clinical need to overcome the increased early non relapse mortality (NRM) associated with delayed neutrophil recovery following cord blood transplant (CBT). Therefore we established a methodology using Notch ligand (Delta1) as a strategy for increasing the absolute number of marrow repopulating CB hematopoietic stem/progenitor cells (HSPC). We previously reported preliminary results of the first 10 patients in 2010 demonstrating the ability of Notch-expanded CB HSPC to provide rapid myeloid recovery post-CBT.1 Herein we present the updated results on 23 patients accrued to this trial aimed at assessment of efficacy as well as the feasibility of overnight shipment of the expanded cell product to three outside institutions. Methods Between July 2006 and March 2013, 23 patients with hematologic malignancies were enrolled in this prospective multi-center Phase I trial coordinated by the Fred Hutchinson Cancer Research Center in which one CB unit was ex vivo expanded prior to infusion. Conditioning consisted of Fludarabine (75mg/m2), Cyclophosphamide (120mg/kg) and TBI (13.2 Gy) over 8 days. On day 0, the unmanipulated CB unit was infused first followed 4 hours later by infusion of the freshly harvested expanded CB cells. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine and MMF beginning on day -3. All CB grafts were 4-6/6 HLA-matched (A/B antigen level, DRB1 allele level) to the recipient. Engraftment, NRM, relapse and GVHD were calculated using cumulative incidence rates to accommodate competing risks. Overall survival was analyzed using Kaplan-Meier estimates. Results Patient diagnosis was AML (n=16), ALL (n=5) and biphenotypic leukemia (n=2). Nine patients (39%) were ≥CR2 and 5 were MRD+ at the time of transplant. Median age was 28 years (range, 4-43) and weight 70 kg (range, 16-91) with a median follow-up of 614 days (range, 271-2443). 22 patients received the expanded graft with one product not meeting release criteria. The cell doses infused were significantly higher in the expanded CB graft: 2.7 (1.5-6.3) vs 6.9 (0.4-27.6) x107 TNC/kg, p<0.0008; 0.15 (0.02-0.57) vs 7.7 (0.62-49.5) x106 CD34/kg, p<0.0001. HLA-matching and ABO incompatibility of the expanded and unmanipulated products were similar. The incidence of neutrophil recovery was 95% (95% CI, 71-100) at a median of 13 days (range, 6-41 days) among the 22 patients receiving expanded CB cells which is significantly faster than that observed in 40 recipients of two unmanipulated units otherwise treated identically at a median time of 25 days (range, 14 to 45; p<0.0001). The incidence of platelet recovery (>20 x 10^9/L) was 77% (CI 95%: 53- 89) by day 100 at a median of 38 days (range, 19 – 134). There was one case of primary graft failure. Importantly, rate of neutrophil recovery correlated with CD34+ cell dose/kg with 8 out of 11 patients receiving greater than 8x106 CD34+cells/kg achieved an ANC ≥ 500/µl within 10 days. 21 patients were evaluable for in vivo persistence of the expanded cells. Ten (48%) demonstrated in vivo persistence beyond one month post infusion. The expanded cell graft was persistent at day 180 in 7 patients, and in those that survived to one year, dominance of the expanded cell graft persisted in one patient. The incidences of grade II-IV and III-IV acute GVHD was 77% (95% CI, 53-89) and 18% (95% CI, 5-36%), respectively; mild chronic GVHD was observed in 4 patients and severe chronic GVHD in one. Probability of OS was 62% (95% CI, 37-79%) at 4 years. Notably, the cumulative incidence of NRM at day 100 was 8% (95% CI, 14-24%) and at 4 years was 32% (95% CI, 8-40%). Nine patients died at a median time of 216 days (range, 31-1578 days) with respiratory failure/infection the most common cause (n=6). There were two relapses at day 156 and 365 post-transplant, with one death due to relapse. Secondary malignancy and primary graft failure were the other 2 causes of death. Conclusions Infusion of Notch-expanded CB progenitors is safe and effective, significantly reducing the time to neutrophil recovery and risks of NRM during the first 100 days. An advantage for infusion of higher numbers of CD34+ cells/kg further demonstrates the need to develop methods that reproducibly provide even greater expansion of repopulating cells than currently achieved to improve efficacy and potentially cost effectiveness. 1. Delaney C, et al, Nat Med. 2010 Feb;16(2):232-6. Disclosures: Delaney: Novartis: DSMB, DSMB Other; Biolife: Membership on an entity’s Board of Directors or advisory committees; medac: Research Funding. Wagner:Novartis: Research Funding; cord use: Membership on an entity’s Board of Directors or advisory committees.

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Blockade of PD-1 in Combination with Dendritic Cell/Myeloma Fusion Cell Vaccination Following Autologous Stem Cell Transplantation Is Well Tolerated, Induces Anti-Tumor Immunity and May Lead to Eradication of Measureable Disease

Blood ◽

10.1182/blood.v126.23.4218.4218 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4218-4218 ◽

Cited By ~ 7

Author(s):

Jacalyn Rosenblatt ◽

Irit Avivi ◽

Noam Binyamini ◽

Lynne Uhl ◽

Poorvi Somaiya ◽

...

Keyword(s):

Stem Cell ◽

Dendritic Cell ◽

Stem Cell Transplantation ◽

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

Advisory Committees ◽

Post Transplant ◽

Fusion Cell ◽

Equity Ownership

Abstract Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to incorporate immunotherapy in an effort to target residual disease. Our group has developed a cancer vaccine in which dendritic cells (DCs) are fused to autologous tumor cells resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. A fundamental challenge to developing a more effective tumor vaccine is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Up-regulation of the PD-1/PDL1 pathway represents a key element contributing to tumor-mediated tolerance, and potentially muting response to vaccination. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (Pidilizumab, MDV9300) in combination with a dendritic cell/myeloma fusion cell vaccine following autologous transplantation. 22 patients have been treated with post-transplant immunotherapy. Mean age was 64. MM cells were isolated from bone marrow and were identified by expression of CD38 or CD138. Mean tumor cell yield was 118x106 cells. Adherent mononuclear cells were isolated from leukapheresis collections and cultured with GM-CSF and IL-4 for 5-7 days, then exposed to TNFα for 48-72 hours to generate mature DCs. DCs expressed co-stimulatory (mean CD86 75%) and maturation markers (mean CD83 50%). DC and MM cells were co-cultured with PEG and fusion cells were quantified by determining the percentage of cells that co-express unique DC and myeloma antigens. Mean fusion efficiency was 41% and the mean cell dose generated was 4 x 106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 92%, 89%, and 85%, respectively. As a measure of their potency as antigen presenting cells, DC/MM fusions potently stimulate allogeneic T cell proliferation ex-vivo (Mean stimulation index of 1.9, 9.2 and 7.1 for tumor, DC and DC/myeloma fusions respectively, n=21) Post-transplant immunotherapy was initiated after recovery from transplant-related toxicities. Median time from transplant to initiation of post-transplant immunotherapy was 80 days. Patients received 3 doses of Pidilizumab at 6-week intervals. DC/myeloma fusion cells vaccination is administered 1 week before each dose of Pidilizumab. To date, 22 patients have completed vaccinations and Pidilizumab. Adverse events judged to be potentially treatment related included grade 1-2 diarrhea, arthralgias, myalgias, fatigue, headache, nausea, chills, transaminitis, cytopenia, elevated TSH, and vaccine site reactions. A significant increase in circulatingtumor reactive lymphocytes was noted following post-transplant immunotherapy, as determined by T cell expressionof IFN-γ by CD8 cells following ex-vivo co-culture withautologous myeloma cell lysate. Mean percentage of tumor reactiveCD8 cells increased from 1.8% post-transplant to a peak of 9.16% following immunotherapy. In the post-transplant period, regulatory T cells fell to minimal levels and remained low throughout the period of immunotherapy. 6 patients achieved a best response of VGPR, 6 patients have achieved a nCR/CR, including 3 who converted to CR following immunotherapy. Median PFS from transplant is 19 months with ongoing follow up. In summary, DC/MM fusion cell vaccination in conjunction with PD1 blockade following ASCT was well tolerated, potently induced anti-tumor immunity, and in a subset of patients, resulted in the eradication of post-transplant measurable disease. Disclosures Richardson: Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Anderson:Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership. Rowe:BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BioLineRx Ltd.: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.

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Comparative Analysis of Independent Ex Vivo functional Drug Screens Identifies Predictive Biomarkers of BCL-2 Inhibitor Response in AML

Blood ◽

10.1182/blood-2018-99-111916 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2763-2763 ◽

Cited By ~ 1

Author(s):

Brian S. White ◽

Suleiman A. Khan ◽

Muhammad Ammad-ud-din ◽

Swapnil Potdar ◽

Mike J Mason ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Research Funding ◽

Drug Response ◽

Ex Vivo ◽

Predictive Performance ◽

Data Sets ◽

Advisory Committees ◽

Data Set ◽

Equity Ownership

Abstract Introduction: Therapeutic options for patients with AML were recently expanded with FDA approval of four drugs in 2017. As their efficacy is limited in some patient subpopulations and relapse ultimately ensues, there remains an urgent need for additional treatment options tailored to well-defined patient subpopulations to achieve durable responses. Two comprehensive profiling efforts were launched to address this need-the multi-center Beat AML initiative, led by the Oregon Health & Science University (OHSU) and the AML Individualized Systems Medicine program at the Institute for Molecular Medicine Finland (FIMM). Methods: We performed a comparative analysis of the two large-scale data sets in which patient samples were subjected to whole-exome sequencing, RNA-seq, and ex vivo functional drug sensitivity screens: OHSU (121 patients and 160 drugs) and FIMM (39 patients and 480 drugs). We predicted ex vivo drug response [quantified as area under the dose-response curve (AUC)] using gene expression signatures selected with standard regression and a novel Bayesian model designed to analyze multiple data sets simultaneously. We restricted analysis to the 95 drugs in common between the two data sets. Results: The ex vivo responses (AUCs) of most drugs were positively correlated (OHSU: median Pearson correlation r across all pairwise drug comparisons=0.27; FIMM: median r=0.33). Consistently, a samples's ex vivo response to an individual drug was often correlated with the patient's Average ex vivo Drug Sensitivity (ADS), i.e., the average response across the 95 drugs (OHSU: median r across 95 drugs=0.41; FIMM: median r=0.58). Patients with a complete response to standard induction therapy had a higher ADS than those that were refractory (p=0.01). Further, patients whose ADS was in the top quartile had improved overall survival relative to those having an ADS in the bottom quartile (p<0.05). Standard regression models (LASSO and Ridge) trained on ADS and gene expression in the OHSU data set had improved ex vivo response prediction performance as assessed in the independent FIMM validation data set relative to those trained on gene expression alone (LASSO: p=2.9x10-4; Ridge: p=4.4x10-3). Overall, ex vivo drug response was relatively well predicted (LASSO: mean r across 95 drugs=0.62; Ridge: mean r=0.62). The BCL-2 inhibitor venetoclax was the only drug whose response was negatively correlated with ADS in both data sets. We hypothesized that, whereas the predictive performance of many other drugs was likely dependent on ADS, the predictive performance of venetoclax (LASSO: r=0.53, p=0.01; Ridge: r=0.63, p=1.3x10-3) reflected specific gene expression biomarkers. To identify biomarkers associated with venetoclax sensitivity, we developed an integrative Bayesian machine learning method that jointly modeled both data sets, revealing several candidate biomarkers positively (BCL2 and FLT3) or negatively (CD14, MAFB, and LRP1) correlated with venetoclax response. We assessed these biomarkers in an independent data set that profiled ex vivo response to the BCL-2/BCL-XL inhibitor navitoclax in 29 AML patients (Lee et al.). All five biomarkers were validated in the Lee data set (Fig 1). Conclusions: The two independent ex vivo functional screens were highly concordant, demonstrating the reproducibility of the assays and the opportunity for their use in the clinic. Joint analysis of the two data sets robustly identified biomarkers of drug response for BCL-2 inhibitors. Two of these biomarkers, BCL2 and the previously-reported CD14, serve as positive controls credentialing our approach. CD14, MAFB, and LRP1 are involved in monocyte differentiation. The inverse correlation of their expression with venetoclax and navitoclax response is consistent with prior reports showing that monocytic cells are resistant to BCL-2 inhibition (Kuusanmäki et al.). These biomarker panels may enable better selection of patient populations likely to respond to BCL-2 inhibition than would any one biomarker in isolation. References: Kuusanmäki et al. (2017) Single-Cell Drug Profiling Reveals Maturation Stage-Dependent Drug Responses in AML, Blood 130:3821 Lee et al. (2018) A machine learning approach to integrate big data for precision medicine in acute myeloid leukemia, Nat Commun 9:42 Disclosures Druker: Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Celgene: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Patient True Talk: Consultancy; Millipore: Patents & Royalties; Monojul: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Bristol-Meyers Squibb: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; ARIAD: Research Funding; Novartis Pharmaceuticals: Research Funding. Heckman:Orion Pharma: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Tyner:AstraZeneca: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Leap Oncology: Equity Ownership; Seattle Genetics: Research Funding; Syros: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Aptose: Research Funding; Agios: Research Funding. Aittokallio:Novartis: Research Funding. Wennerberg:Novartis: Research Funding.

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Combining Next Generation Proteomics Platforms with Drug Sensitivity Resistance Testing AllowsÂ Identification of Physiologically Distinct Sub-ClonesÂ That Can Inform Therapeutic and Drug Development Strategies

Blood ◽

10.1182/blood-2018-99-115910 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1901-1901

Author(s):

Despina Bazou ◽

Muntasir M Majumder ◽

Ciara Tierney ◽

Sinead O'Rourke ◽

Pekka Anttila ◽

...

Keyword(s):

Mass Spectrometry ◽

Board Of Directors ◽

Research Funding ◽

Drug Sensitivity ◽

Plasma Cells ◽

Ex Vivo ◽

Resistance Testing ◽

Proteomics Analysis ◽

Advisory Committees ◽

Group I

Abstract Introduction: A hallmark of Multiple Myeloma (MM) is the sequel development of drug resistant phenotypes, which may be present initially or emerge during the course of treatment. These drug resistant phenotypes reflect the intra-tumor and inter-patient heterogeneity of this cancer. Most MM cells are sensitive to proteasome inhibitors (PIs), which have become the standard of care in the treatment of newly diagnosed and relapsed MM. However, resistance develops (intrinsic/acquired). Although several novel drugs have recently been approved or are in development for MM, there are few molecular indicators to guide treatment selection. To address this limitation we have combined mass spectrometry-based proteomics analysis together with ex vivo drug response profiles and clinical outcome to elucidate a best possible accurate phenotype of the resistant sub-clones, thus yielding a theranostic profile that will inform therapeutic and drug development strategies. Methods: We performed mass spectrometry-based proteomics analysis on plasma cells isolated from 38 adult MM patient bone marrow aspirates (CD138+). Samples were obtained at diagnosis or prior to commencing therapy. The participating subjects gave written informed consent in accordance with the Declaration of Helsinki that was approved by local ethics committees. For the proteomics analysis, peptides were purified using the filtered aided sample preparation (FASP) method. Subsequently, samples were prepared for label-free liquid chromatography mass spectrometry (LC-MS/MS) using a Thermo Scientific Q-Exactive MS mass spectrometer. Proteins were analysed using the MaxQuant and Perseus software for mass-spectrometry (MS)-based proteomics data analysis, UniProtKB-Swiss Prot database and KEGG Pathway database. In parallel, we undertook a comprehensive functional strategy to directly determine the drug dependency of myeloma plasma cells based on ex vivo drug sensitivity and resistance testing (DSRT)as previously described (1). Results: Our initial proteomic analysis was generated by examining MM patient plasma cells, grouped based on DSRT to 142 anticancer drugs including standard of care and investigational drugs. Each of the 142 drugs was tested over a 10,000-fold concentration range, allowing for the establishment of accurate dose-response curves for each drug in each patient. MM patients were stratified into four distinct subgroups as follows: highly sensitive (Group I), sensitive (Group II), resistant (Group III) or highly resistant (Group IV) to the panel of drugs tested. We then performed blinded analysis on the 4 groups of CD138+ plasma cells divided based on the ex vivo sensitivity profile, identifying a highly significant differential proteomic signature between the 4-chemosensitivity profiles, with Cell Adhesion Mediated-Drug Resistance (CAM-DR) related proteins (e.g. integrins αIIb and β3) significantly elevated in the highly resistant phenotype (Group IV). In addition our results showed that Group I patients displayed significant upregulation of cell proliferation proteins including: MCM2, FEN1, PCNA and RRM2. Furthermore, Group I patients have shorter Progression Free Survival (PFS) as well as Overall Survival (OS) compared to the other subgroups. Figure 1 shows the Heatmap summarizing the expression of proteins (log2 fold change) in the four distinct MM patient subgroups. Conclusions:Our findings suggest that combining a proteomics based study together with drug sensitivity and resistance testing allows for an iterative adjustment of therapies for patients with MM, one patient at a time, thus providing a theranostic approach. Our results suggest that the disease driving mechanisms in the patient subgroups are distinct, with highly resistant patients exhibiting cell adhesion mediated cytoprotection, while highly sensitive patients show an increased cell proliferation protein profile with shorter PFS and OS. Our study aims to guide treatment decisions for individual cancer patients coupled with monitoring of subsequent responses in patients to measure and understand the efficacy and mechanism of action of the drugs. Future work will include the establishment of flow cytometry-based screening assays to identify the different resistant phenotypes at diagnosis/relapse. References: (1) M. M. Majumder et al., Oncotarget 8(34), 56338 (2017) Disclosures Anttila: Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Silvennoinen:Amgen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Heckman:Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding.

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Preliminary Study of Ruxolitinib and Venetoclax for Treatment of Patients with T-Cell Prolymphocytic Leukemia Refractory to, or Ineligible for Alemtuzumab

Blood ◽

10.1182/blood-2021-149228 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1201-1201

Author(s):

Charles Herbaux ◽

Stéphanie Poulain ◽

Damien Roos-Weil ◽

Jacques-Olivier Bay ◽

Yann Guillermin ◽

...

Keyword(s):

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Advisory Committees ◽

Prolymphocytic Leukemia ◽

Oncology Research ◽

Grade 3 ◽

Stat Pathway

Abstract Background: Ruxolitinib (RUX), a JAK1/JAK2 inhibitor, and venetoclax (VEN), a BCL-2 inhibitor are 2 drug candidates recently identified as promising candidate for the treatment of T-Cell prolymphocytic leukemia (T-PLL). We recently reported that JAK/STAT pathway inhibition with RUX enhances BCL-2 dependence, thereby sensitizing T-PLL cells to VEN (Herbaux et al., Blood, 2021). We also showed that JAK/STAT pathway mutational status could impact RUX activity. Here, we report results on the 15 first patients who were treated with RUX and VEN oral combination for T-PLL. All patients were refractory to, or ineligible for alemtuzumab, the principal therapeutic option to date. Methods: In this multicenter retrospective study from the French Innovative Leukemia Organization, 15 patients with T-PLL (according to consensus criteria) were included. All patients were informed about the off-label use of this combination and provided informed consent. Patients received a maximum dose of RUX 15 mg twice daily, and VEN 800 mg daily. VEN was started with daily ramp-up from 20 mg to 800 mg over 6 days, with TLS prophylaxis (rasburicase and IV hydration). Responses were assessed by consensus criteria. Next generation sequencing (NGS) was performed using a custom-designed panel of 33 genes, including among others: ATM, TP53, IL2R, JAK1, JAK3, and STAT5B. CytoScan HD microarray (Affymetrix) were used to study copy number variation and or uniparental disomy. In vivo dynamic BH3 profiling (DBP) was performed on samples obtained from two patients on treatment. Results: All 15 patients were refractory or relapsing after chemotherapy (mostly bendamustine and pentostatin), except one. They were either refractory to (n=10) or ineligible (n=5) for alemtuzumab (ineligibility was decided by the treating physician based on age and comorbidities). The median age was 70 years (48-88). Within a week of starting RUX, a transient increase of the absolute lymphocyte count was observed in 66.6% of the patients. Based on the molecular status of the JAK/STAT pathway, we established 2 groups of patients. One with samples where no mutations were found (WT, n=3), and one with at least one mutation in the JAK/STAT pathway (MUT, n=12). The overall response rate (ORR) was 73.3%, with only partial responses. Five patients nearly fulfilled CR criteria except that they had persistent lymphocytosis (over 4 x 10 9/L), all of them were in the MUT group. ORR was 83.3% in the MUT group, and only one patient of the WT group obtained a PR. With a median follow-up of 73 days (22 to 368), the median progression free survival was significantly shorter in the WT group in comparison to the MUT group (1.8 months versus 5.6 months, p=0.04, Figure). Of note, four patients were treated with VEN monotherapy before the start of the combination with RUX. With that treatment, 3 of these patients achieved stable disease followed by progression within 2 to 3 months, while 1 was primary refractory to VEN monotherapy. The most frequent reported adverse events (AEs) of the RUX plus VEN combination were cytopenias, with 46.6% grade 3 or 4 thrombocytopenia and 40% grade 3 or 4 neutropenia. DBP showed that overall priming and BCL2 dependence increased in vivo (n=2) during the treatment with RUX and VEN. Finally, SNP arrays identified clonal evolution in the 3 patients evaluated sequentially (before treatment versus at progression). In one case, emergence of EZH2 and JAK1 mutation was also observed at progression using NGS. Conclusions: These preliminary results suggest promising activity of RUX plus VEN in T-PLL, and justify the development of a prospective clinical trial of this combination. Our data seem to show that this combination may be especially active for patients with JAK/STAT pathway activating mutations and that disease progression is associated with clonal evolution. Updated results will be presented at the meeting. Figure 1 Figure 1. Disclosures Herbaux: Janssen: Honoraria; Roche: Honoraria; Abbvie: Honoraria, Research Funding; Takeda: Honoraria, Research Funding. Lemonnier: Gilead: Other: travel grant; Institut Roche: Research Funding. Laribi: Jansen: Research Funding; AstraZeneca: Other: Personal Fees; Takeda: Other: Personal Fees, Research Funding; Novartis: Other: Personal Fees, Research Funding; Astellas Phama, Inc.: Other: Personal Fees; IQONE: Other: Personal Fees; AbbVie: Other: Personal Fees, Research Funding; Le Mans Hospital: Research Funding; BeiGene: Other: Personal Fees. Moreaux: Diag2Tec: Consultancy. Morschhauser: Janssen: Honoraria; Servier: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; AstraZenenca: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria; Genentech, Inc.: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genmab: Membership on an entity's Board of Directors or advisory committees. Davids: Ascentage Pharma: Consultancy, Research Funding; MEI Pharma: Consultancy, Research Funding; Merck: Consultancy; Eli Lilly and Company: Consultancy; Adaptive Biotechnologies: Consultancy; Pharmacyclics: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Research to Practice: Consultancy; BeiGene: Consultancy; Surface Oncology: Research Funding; Verastem: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Takeda: Consultancy; Astra-Zeneca: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy; AbbVie: Consultancy; Genentech: Consultancy, Research Funding; Janssen: Consultancy; MEI Pharma: Consultancy. Ysebaert: Abbvie, AstraZeneca, Janssen, Roche: Other: Advisory Board, Research Funding. OffLabel Disclosure: Ruxolitinib and venetoclax are used offlabel for patients refractory to current therapeutic options, based on preclinical data.

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CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Blood ◽

10.1182/blood-2018-99-118926 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3199-3199 ◽

Cited By ~ 2

Author(s):

Subhashis Sarkar ◽

Sachin Chauhan ◽

Arwen Stikvoort ◽

Alessandro Natoni ◽

John Daly ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Advisory Committees ◽

Cd38 Expression ◽

Rpmi 8226

Abstract Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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