Overactivation of the NLRP3 Inflammasome in Chronic Myelomonocytic Leukemia KRAS Mutated Patients Can be Detected By the Apoptosis-Associated Speck-like Protein (ASC) and Reverted By IL1β Inhibitors

Abstract It has been recently shown that RAS mutations, which occur in 11-38% of Chronic Myelomonocytic Leukemia (CMML), do not only act via RAS/MEK/ERK signaling, but contribute to the disease through NLRP3 inflammasome activation (Hamarsheh, Nat Comm 2020). Despite a therapeutic approach based on NLRP3/IL1β axis blockade, as bring to a stem cell transplantation (SCT) has been proposed, data on the efficacy of IL1β inhibitors in hematopoietic neoplasms is limited. A 55 year old man with previous autoinflammatory episodes (constrictive pericarditis) was diagnosed on September 2020 of CMML-1 KRAS G12D (Inter-2). Due to worsening (orchiepidedymitis, pneumonitis, cellulitis), and the impossibility of performing an SCT at that time, on December 02 2020 he started anakinra (a IL1β receptor antagonist) with good response. Due to new episodes of autoinflammation, anakinra was discontinued (12 April 2021) with severe clinical worsening (heart failure) and no response to diuretic/corticosteroid. After anakinra was restarted (04 May 2021), a progressive improvement was seen, allowing a successful pericardiectomy before an SCT. We obtained blood samples from this patient (at different times) and plasma and whole blood samples from 11 and 5 other CMML KRAS mut patients, respectively. We also included CMML patients without KRAS mutations (KRAS wt) (n=8), with sepsis (n=5) and healthy individuals (n=9). Plasma levels of 15 inflammatory cytokines associated with NLRP3 inflammasome and NFkB pathways were measured using a customized MILLIPLEX ® kit. The inflammasome marker activation assays were conducted as previously published (Martínez García JJ, Nature Comm 2019). Compared to healthy controls, KRAS wt CMML patients did not show differences in any cytokine tested, except IL6, while KRAS mut patients showed significantly higher levels of IL1α, IL1ra, IL18, IL12p40 (associated with NLRP3 inflammasome), IL6, IL8 (associated with NFkB pathway) and M-CSF (Fig. 1A B). Compared to KRAS wt CMML patients, those with KRAS mut showed higher levels of cytokines associated with both the NLRP3 and NFkB pathways, reaching statistical significance for those related with NLRP3 inflammasome. We also observed changes in inflammasome related cytokines before and after anakinra (Table 1). This cytokine profile in the plasma made us analyze the oligomerization of ASC as a marker of inflammasome activation in monocytes of KRAS mut CMML. We found that in all cases of KRAS mut CMML patients around 30 to 80% of monocytes presented oligomers of ASC measured by the time of flight assay, while in healthy donors and KRAS wt CMML patients, ASC oligomerization occurred upon NLRP3 inflammasome activation with lipopolysaccharide (LPS) + ATP or Pyrin inflammasome activation with LPS and Clostridium difficile B toxin (TcdB) (Fig. 2A). Ex vivo activation of PBMCs from KRAS mut CMML patients showed that despite the high percentage of cells with ASC oligomers, very low levels of IL1b released from these cells, even when NLRP3 was activated with LPS+ATP (Fig. 2B), suggesting that this inflammasome is activated in vivo and could not be further activated ex vivo. As control, Pyrin inflammasome activation in PBMCs from KRAS mut CMML was able to induce IL1b release similarly to healthy controls (Fig. 2B). We then found that anakinra treatment of the KRAS mut CMML patient followed in this study, resulted in a decrease of the percentage of monocytes with basal active inflammasomes (Fig. 2C). A little ex vivo activation of the NLRP3 inflammasome was obtained when cells were treated with LPS+ATP, while Pyrin inflammasome was activated at normal levels after LPS+TcdB treatment (Fig. 2D). The inflammasome basal activation increased in the monocytes of the KRAS mut CMML patient after anakinra withdraw and during clinical deterioration and restarting anakinra (second arrow) decreased the basal percentage of monocytes with ASC oligomers (Fig. 2C). Since ASC oligomers are associated to pyroptosis via caspase 1 activation and gasdermin D processing, we then analyzed pyroptotic markers in the plasma of the patient during the time. ASC was increased when monocytes presented elevated percentage of ASC oligomers (Fig. 2E), suggesting that ASC detection could be a promising biomarker. Overall, we show that, in vivo, the NLRP3 inflammasome activation of KRAS mut CMML patients may revert with IL1β blockers. ASC could identify those candidates to receive this therapy. PI18/00316 Figure 1 Figure 1. Disclosures Jerez: Novartis: Consultancy; BMS: Consultancy; GILEAD: Research Funding. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau. Hernández-Rivas: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ferrer Marin: Cty: Research Funding; Incyte: Consultancy, Research Funding; Novartis: Speakers Bureau.

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Phase Ib Study of Oral Panobinostat In Combination with 5-Azacitidine (5-aza) In Patients with Myelodysplastic Syndromes (MDS), Chronic Myelomonocytic Leukemia (CMML), or Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v116.21.4957.4957 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4957-4957 ◽

Cited By ~ 1

Author(s):

Pierre Fenaux ◽

Daniel J DeAngelo ◽

Guillermo Garcia-Manero ◽

Michael Lübbert ◽

Anand P. Jillella ◽

...

Keyword(s):

Febrile Neutropenia ◽

Adverse Events ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Chronic Myelomonocytic Leukemia ◽

Advisory Committees ◽

Dose Cohort ◽

Phase Ib ◽

Myelomonocytic Leukemia

Abstract Abstract 4957 Background: Panobinostat is a potent pan-deacetylase inhibitor (pan-DACi) that causes increased acetylation of target proteins such as HSP90, p53, α-tubulin and HIF-1α which are involved in cell cycle regulation, gene transcription, angiogenesis, and tumor cell survival. Preliminary evidence from phase I trials has demonstrated anti-tumor activity in patients with hematologic malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The advent of hypomethylating agents, such as 5-aza, represent a significant advancement in the treatment of MDS, chronic myelomonocytic leukemia (CMML), and AML. Although an improvement in clinical outcomes has been observed, including increased overall survival in patients with MDS, a substantial number of patients do not benefit from the therapies currently available. Preclinical studies suggest that the combination of a demethylating agent and a pan-DACi represents a rational strategy to reverse silencing of tumor suppressor genes, which contributes to the malignant phenotype, and improve outcomes in patients with MDS and AML. In this study, the combination of the pan-DACi, panobinostat, and the hypomethylating agent, 5-aza, was evaluated in patients with MDS, CMML and AML. Methods: This phase Ib, open-label, multicenter, dose-finding study is comprised of 2 stages: a dose-escalation stage to determine the maximum tolerated dose (MTD) of panobinostat in combination with standard dose 5-aza, and a subsequent expansion stage to evaluate safety, tolerability, and preliminary activity at the MTD dose level. The primary endpoint is incidence of dose-limiting toxicity (DLT) and secondary endpoints include type, duration, frequency, and relationship of adverse events (AEs) to the combination. Exploratory endpoints include clinical response and hematologic improvement according to IWG response criteria, and biomarker analysis of methylation status and expression of disease-associated genes in peripheral blood cells prior to and during therapy. Adult patients with IPSS INT-2 or high-risk MDS, CMML, or AML with multi-lineage dysplasia and ≤ 30% marrow blasts who are candidates for therapy with 5-aza and have not received a prior hypomethylating agent or pan-DACi are eligible for enrollment on the trial. Oral panobinostat was administered on Days (D) 3, 5, 8, 10, 12, and 15, starting at 20 mg, in combination with 5-aza (75 mg/m2 sc D 1–7) during a 28-D cycle. Patients received treatment for ≤ 6 cycles or until progression of disease, incidence of unacceptable toxicity, or withdrawal of consent. Results: To date, 11 patients have been enrolled including 9 patients with MDS, 1 patient with AML and 1 patient with CMML. The median age of patients enrolled on the trial was 69.0 (60-80). Patients have been evaluated at 2 panobinostat dose cohorts; 6 (20 mg) and 5 (30 mg). The AE analysis is based on 9 patients (6 from 20 mg cohort and 3 from 30 mg cohort) and the nature and incidence of AEs observed in the two cohorts were similar. Adverse events regardless of study drug relationship included nausea (4 [44%]), vomiting, fatigue (5 [55%] each) and asthenia (3 [33%]). Grade 3/4 AEs suspected to be treatment related included thrombocytopenia (2 [22%], febrile neutropenia and arthritis (1 [11%] each). Serious adverse events observed included febrile neutropenia, asthenia (2 [22%] each), atrial fibrillation and septic shock (1 [11%] each). One DLT has been observed (grade 4 febrile neutropenia) in the 20 mg panobinostat dose cohort. Conclusions: Panobinostat has been well tolerated up to a dose of 30 mg in combination with 5-aza (75 mg/m2) with dose escalation ongoing. Patients are currently being enrolled at the 40mg dose cohort. The most common AEs observed included febrile neutropenia, thrombocytopenia with one DLT observed (grade 4 febrile neutropenia) in the 20mg panobinostat dose cohort. The current data show that the addition of panobinostat to 5-aza is safe with no unexpected toxicities. Updated data, including safety and preliminary efficacy data will be presented at the meeting. Disclosures: Fenaux: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding. Off Label Use: Panobinostat is an investigational agent currently being evaluated for the treatment of hematologic and solid malignancies. DeAngelo: Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Winiger: Novartis Pharma AG: Employment. Squier: Novartis: Employment. Li: Novartis: Employment. Ottmann: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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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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Transition of Chronic Myeloid Leukemia to Chronic Myelomonocytic Leukemia As a Tool to Observe Development of Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood.v126.23.5223.5223 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5223-5223

Author(s):

Jamshid S Khorashad ◽

Srinivas K Tantravahi ◽

Dongqing Yan ◽

Anna M. Eiring ◽

Hannah M. Redwine ◽

...

Keyword(s):

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Clonal Evolution ◽

Chronic Myelomonocytic Leukemia ◽

Imatinib Treatment ◽

Advisory Committees ◽

Clonal Architecture ◽

Myelomonocytic Leukemia

Abstract Introduction. Development of abnormal Philadelphia (Ph) negative clones following treatment of chronic myeloid leukemia (CML) patients with imatinib has been observed in 3 to 9% of patients. Here we report on a 77 year old male diagnosed with CML that responded to imatinib treatment and subsequently developed chronic myelomonocytic leukemia (CMML). He achieved major cytogenetic response within 3 months but this response coincided with the emergence of monocytosis diagnosed as CMML. Five months after starting imatinib treatment the patient succumbed to CMML. We analyzed five sequential samples to determine whether a chronological order of mutations defined the emergence of CMML and to characterize the clonal evolution of the CMML population. Materials and Method. Five samples (diagnostic and four follow up samples) were available for analysis. CMML mutations were identified by whole exome sequencing (WES) in CD14+ cells following the onset of CMML, using CD3+ cells as constitutional control. Mutations were validated by Sequenom MassARRAY and Sanger sequencing and quantified by pyrosequencing. Deep WES was performed on the diagnostic sample to determine whether the mutations were present at CML diagnosis. To determine the clonal architecture of the emerging CMML, colony formation assays were performed on the diagnostic and the next two follow-up samples (Samples 1-3). More than 100 colonies per sample were plucked for DNA and RNA isolation. The DNA from these colonies were tested for the presence of the confirmed CMML mutations and the RNA was used for detection of BCR-ABL1 transcript using a Taqman real time assay. Results. Four mutations were identified by Sequenom and WES throughout the patient's time course [KRASG12R, MSLNP462H, NTRK3V443I and EZH2I669M ]. Sequenom did not identify these at diagnosis while deep WES did. Clones derived from colony formation assay revealed three distinct clones present in all samples analysed. Clone 1 had only KRASG12R, clone 2 had KRASG12R, MSLNP462H, and NTRK3V443I, and clone 3 had all four mutations. All clones containing any of these four mutations were BCR/ABL1 negative. Analysis of clonal architecture indicated that KRASG12R was acquired first and EZH2I669M last, while MSLNP462H and NTRK3V443I were acquired in between. These CMML clones increased proportionately as clinical CML metamorphosed into clinical CMML after initiation of imatinib therapy. Consistent with the colony data, pyrosequencing revealed that the ratio between the mutants remained largely stable throughout the follow up period. Conclusion. This case illustrates how targeted therapy impacts clonal competition in a heterogeneous MPN. While the CML clone was dominant in the absence of imatinib, it was quickly outcompeted by the CMML clones upon initiation of imatinib therapy. The clonal architecture analysis, in combination with in vivo kinetics data, suggest that the KRASG12R mutation alone was able to produce a CMML phenotype as clones with just KRASG12R remained at a relatively stable ratio during follow up. Unexpectedly, acquisition of additional mutations, including EZH2I669M as the last mutational event identified in this patient, did not increase clonal competitiveness, at least in the peripheral blood. These data show that clonal evolution may not invariably increase clonal fitness, suggesting that factors other than Darwinian pressures contribute to clonal diversity in myeloproliferative neoplasms. Disclosures Deininger: Gilead: Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Characteristics and Outcomes Of Patients (pts) With Multiple Myeloma (MM) Who Develop Therapy (t)-Related Myelodysplastic Syndrome (MDS), t-Chronic Myelomonocytic Leukemia (CMML), Or t-Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v122.21.1424.1424 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1424-1424

Author(s):

Naveen Pemmaraju ◽

Dhaval Shah ◽

Hagop M Kantarjian ◽

Verena Wagner ◽

Robert Z. Orlowski ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Chemotherapeutic Agents ◽

Chronic Myelomonocytic Leukemia ◽

Advisory Committees ◽

Myelomonocytic Leukemia ◽

Acute Myeloid

Abstract Background There has been a significant improvement in the outcome for patients (pts) with MM over the last decade, mainly due to the availability of immunomodulatory (IMiD) drugs and proteasome inhibitors (PI). The improvement in survival has also increased the risk of second primary malignancies (SPM), such as therapy-related myelodysplastic syndrome (t-MDS), therapy-related chronic myelomonocytic leukemia (t-CMML) or therapy-related acute myeloid leukemia (t-AML). However, little is known about the characteristics and outcomes of pts with t-MDS, t-CMML or t-AML. Methods We aimed to study the characteristics and outcome of pts who developed t-MDS, t-AML and t-CMML as SPM after the treatment of MM. We reviewed our database of pts with MM who were treated at our institution between 1993 and 2011. We identified 49 pts who were diagnosed to have t-MDS, t-CMML, or t-AML. The primary objective of this study was to evaluate the time to develop t-MDS, t-AML and t-CMML, their response to treatment and overall survival. Results Median age of pts at diagnosis of MM was 61 years. Forty-seven (96%) pts had symptomatic MM, while 2 (4%) had asymptomatic myeloma. Forty-seven (95%) pts with symptomatic myeloma received systemic therapy. Eleven (22%) pts were treated with IMiD or PI: lenalidomide 3, thalidomide 6 and bortezomib 2. Thirty-eight (78%) pts were treated with various conventional chemotherapeutic agents including melphalan, cyclophosphamide, doxorubicin, vincristine, etoposide, cisplatin, idarubicin, thiotepa, busulfan, carmustine and cytarabine. Fourteen (28%) pts also received radiation therapy to the affected areas. Twenty (41%) pts underwent high-dose chemotherapy and autologous hematopoietic stem cell transplantation (auto-HCT). Fourteen pts received maintenance therapy after auto-HCT with either thalidomide, lenalidomide, dexamethasone or bortezomib. Median time from the diagnosis of MM to t-MDS, t-CMML or t-AML was 6 years [0 – 24]. Thirty-four (69 %) pts developed t-MDS, 12 (24%) t-AML, and 3 (6%) t-CMML. Median age at diagnosis of t-MDS, t- CMML, or t-AML was 65 years. Twenty-seven (79%) pts with t-MDS and all 12 pts with t-AML had complex/high risk cytogenetics. Most common cytogenetic abnormalities involved chromosome 5 and 7. Thirty four (69%) pts received at least 1 cycle of induction chemotherapy either with conventional chemotherapeutic agents or investigational drugs. Only 9 pts (26%) achieved complete remission (CR). Median duration of CR in these pts was 4 months [1 – 62]. Median overall survival (OS) of pts who received induction therapy was 6.0 months [0-30]. Five (11%) pts received an allogeneic stem cell transplant with three achieving CR. Median OS in this subgroup of pts was 18 months [9 – 23]. Median OS for all 49 pts after diagnosis of t-MDS, t-CMML or t-AML was 6.0 months [0 – 30] Conclusion Development of t-MDS, t-CMML, or t-AML in pts with MM is associated with a poor outcome. These pts in general have complex cytogenetic abnormalities, chemo-resistant disease, a short CR and OS. A better understanding of disease biology and novel therapeutic approaches are warranted. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees. Qazilbash:Otsuka: Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millennium Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

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Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

Journal of Experimental Medicine ◽

10.1084/jem.20171419 ◽

2017 ◽

Vol 214 (11) ◽

pp. 3219-3238 ◽

Cited By ~ 152

Author(s):

Hua Jiang ◽

Hongbin He ◽

Yun Chen ◽

Wei Huang ◽

Jinbo Cheng ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Ex Vivo ◽

Binding Motif ◽

Inflammasome Activation ◽

Therapeutic Effects ◽

Nlrp3 Inflammasome Activation ◽

Molecule Inhibitor ◽

Synovial Fluid Cells

The NLRP3 inflammasome has been implicated in the pathogenesis of a wide variety of human diseases. A few compounds have been developed to inhibit NLRP3 inflammasome activation, but compounds directly and specifically targeting NLRP3 are still not available, so it is unclear whether NLRP3 itself can be targeted to prevent or treat diseases. Here we show that the compound CY-09 specifically blocks NLRP3 inflammasome activation. CY-09 directly binds to the ATP-binding motif of NLRP3 NACHT domain and inhibits NLRP3 ATPase activity, resulting in the suppression of NLRP3 inflammasome assembly and activation. Importantly, treatment with CY-09 shows remarkable therapeutic effects on mouse models of cryopyrin-associated autoinflammatory syndrome (CAPS) and type 2 diabetes. Furthermore, CY-09 is active ex vivo for monocytes from healthy individuals or synovial fluid cells from patients with gout. Thus, our results provide a selective and direct small-molecule inhibitor for NLRP3 and indicate that NLRP3 can be targeted in vivo to combat NLRP3-driven diseases.

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Hypomethylating Agent Therapy for Chronic Myelomonocytic Leukemia Does Not Impact Acute Myeloid Leukemia Transformation or Survival

Blood ◽

10.1182/blood-2020-142747 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-7

Author(s):

Sandrine Niyongere ◽

Yamini Kathari ◽

Zeba Singh ◽

Emily J. Vannorsdall ◽

Ashkan Emadi ◽

...

Keyword(s):

Clinical Trial ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Chronic Myelomonocytic Leukemia ◽

Institutional Research ◽

Research Support ◽

Advisory Committees ◽

Clinical Trial Research ◽

Myelomonocytic Leukemia

Background: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder with features of both myeloproliferative neoplasm and myelodysplastic syndrome (MDS). CMML is characterized by persistent blood monocytosis >1 x 109/L, bone marrow dysplasia in one or more hematopoietic cell lines, and increased risk of transformation to acute myeloid leukemia (AML). Our review of SEER Medicare data (Haematologica 2013;98:584) demonstrated that, compared to MDS, CMML has shorter overall survival (OS) and more frequent progression to AML. Hypomethylating agents (HMAs) have become standard therapy for CMML, with reported response rates of 37-69%, but their impact on AML transformation and OS is unclear. Methods: We retrospectively reviewed CMML patients treated at the University of Maryland Greenebaum Comprehensive Cancer Center between January 2000 and December 2019. Clinical characteristics, treatments, AML progression, time to AML progression (TTP), and OS were recorded and analyzed. Descriptive statistics were used for baseline characteristics and Kaplan-Meier analysis was performed for time-to-event data. Statistical analyses were performed using GraphPad Prism 8®. Results: We identified 71 patients with CMML, 82% male and 73% white, with a median age of 69 (range 25 - 96) years; 51% had <10% bone marrow (BM) blasts and 45% had low-risk cytogenetic findings (normal karyotype or -Y). Most patients treated prior to 2005 received hydroxyurea and/or erythropoiesis-stimulating agents or were enrolled on clinical trials, while patients treated since 2005 received HMAs as primary therapy. Median follow-up was 41.1 months. The median OS of the entire cohort was 20 months, with 46% of patients progressing to AML with a median TTP of 11.5 months. By the MD Anderson Prognostic Scoring System at time of diagnosis, CMML was low-risk in 24 patients, intermediate-1 in 16, intermediate-2 in 14, and high-risk in 17. Forty-six patients received HMAs, with an overall response rate (ORR) of 54% (complete response or partial response), while 25 patients did not receive HMAs. Patient and disease characteristics were similar in HMA- and non-HMA-treated patients (Table 1). The estimated OS of HMA-treated patients was 20 months, compared to 14 months for non-HMA-treated patients (p =0.43) (Figure 1). AML transformation occurred in 52% of patients treated with HMAs, with TTP ranging from 3 to 65 months, and in 33% patients not treated with HMAs, with TTP ranging from 5 to 47 months. Most patients receiving HMAs (63%) received ≥ 6 cycles; 46% transformed to AML despite initial response, often in a sudden and unpredictable manner. HMAs were azacitidine in 13 patients, decitabine in 24, azacitidine followed by decitabine in 4, and decitabine followed by azacitidine in 5. Five CMML patients in our cohort underwent allogenic stem cell transplantation. Four of the five relapsed with transformation to AML post transplant, and only one patient remains in remission, 9 months post transplant. Conclusions: Despite a 54% ORR, HMA treatment did not have a significant impact on frequency of AML transformation, or OS in our cohort. Based on our data, favorable response rates previously reported with HMAs and also seen in our patients do not appear to translate into decreased frequency of AML transformation or prolonged OS. Though our study is a retrospective study with inherent selection bias, our results underscore the ongoing need for novel therapies and for clinical trials for CMML patients. Disclosures Niyongere: Kartos Therapeutics: Other: Received clinical trial research support with Kartos Therapeutics ; Forty Seven: Other: Received clinical trial research support with Forty Seven. Emadi:Amgen: Membership on an entity's Board of Directors or advisory committees; KinaRx: Other: co-founder and scientific advisor; NewLink Genetics: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding. Doung:Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: clinical trial research support; Incyte: Other: clinical trial research support; Astex: Other: clinical trial research support; MedPacto: Other: clinical trial research support. Baer:Takeda: Other: Institutional research funding; Oscotec: Other: Institutional research funding; Kite: Other: Institutional research funding; Incyte: Other: Institutional research funding; Forma: Other: Institutional research funding; Astellas: Other: Institutional research funding; AbbVie: Other: Institutional research funding.

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T Cells Educated By DC/AML Fusions in the Context of 4-1BB Costimulation As a Potent Strategy for Adoptive Cellular Therapy

Blood ◽

10.1182/blood-2019-131491 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2673-2673

Author(s):

Jessica Liegel ◽

Dina Stroopinsky ◽

Haider Ghiasuddin ◽

Adam Morin ◽

Marzia Capelletti ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Cellular Therapy ◽

Ex Vivo ◽

Advisory Board ◽

Adoptive Cellular Therapy ◽

Advisory Committees ◽

Equity Ownership

Introduction: Our group has developed a novel vaccine using patient-derived acute myeloid leukemia (AML) cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. In a phase I/II clinical trial DC/AML vaccination led to an expansion of leukemia-specific T cells. We hypothesized that the fusion vaccine offered a unique platform for ex vivo expansion of functionally potent leukemia specific T cells with broad specificity targeting shared and tumor specific neoantigens. We postulated that incorporating 4-1BB (CD137) mediated co-stimulation would further enhance activation of antigen specific T cells and the development of a crucial memory response as well as promote survival and persistence. Here we describe therapeutic exploration of the use of 4-1BB to augment vaccine-educated T cells for adoptive cellular therapy in an immunocompetent murine model. Methods: DC/AML fusion vaccine was generated using DCs obtained from C57BL/6J mice and syngeneic C1498 AML cells as previously described. T cells were obtained from splenocytes after magnetic bead isolation and cultured with irradiated DC/AML fusion vaccine in the presence of IL-15 and IL-7. Following co-culture, 4-1BB positive T cells were ligated using agonistic 4-1BB antibody (3H3 clone, BioXCell) and further selected with RatIgG2a magnetic beads (Easy Sep). Subsequently T cells were expanded with anti-CD3/CD28 activation beads (Dynabeads). In vivo, mice underwent retro-orbital inoculation with C1498 and vaccination with irradiated fusion cells the following day. Agonistic mouse anti-4-1BB antibody was injected intraperitoneally on day 4 and day 7. In addition, C1498 cells were transduced with Mcherry/luciferase and a reproducible model of disease progression was established. Results: DC/fusion stimulated T cells showed increased immune activation as measured by multichannel flow cytometric analysis. Compared to unstimulated T cells, there was 5-fold increase in CD4+CD25+CD69+, and a 10-fold and 7-fold increase in 4-1BB and intracellular IFNƔ expression on CD8+ cells respectively. Following agonistic 4-1BB ligation and bead isolation, the proliferation rate was increased in the 4-1BB positive fraction as compared to both 4-1BB negative cells and unstimulated T cells. In addition, the 4-1BB positive fraction demonstrated increased cytotoxicity, as measured by a CTL assay detecting granzyme B with 1:10 tumor to effector cells. A shift from naïve to memory T cell phenotype was also observed. Following DC/fusion stimulation, CD44+CD62L- cells comprised 67% of CD8+ cells versus 20% without stimulation, the latter reflecting the effect of cytokines alone. Following 4-1BB ligation and anti-CD3/CD28 bead expansion, this phenotype was retained with the CD4+ and CD8+ effector memory and central memory compartments comprising the majority of T cells. Such findings are significant as presence of memory T cell populations are a critical component for successful adoptive cell transfer. The effect of agonistic 4-1BB antibody following vaccination was evaluated in vivo in an aggressive immunocompetent murine AML model. The combination of DC/AML fusion vaccine with 4-1BB antibody was associated with increased long-term survival (>120 days) of 40% versus 20% of mice treated with vaccine alone while all controls required euthanasia by 40 days. Conclusion: In the current study we have demonstrated the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo as demonstrated by both early-activation (CD25,CD69), upregulation of antigen-specific markers (CD137) and cytokine secretion. Further enhancement of the cellular product using agonistic 4-1BB ligation and isolation simultaneously enriches for antigen-activated cells, as demonstrated by more potent cytotoxicity, as well as promoting memory phenotype and survival. Use of 4-1BB ligation for antigen-specific selection while providing an agonistic co-stimulatory signal is a potentially novel approach for development of non-engineered T cells. Ongoing experiments evaluating the efficacy of 4-1BB selected vaccine educated T cells using bioluminescence monitoring will be reported as well as in vitro use of patient-derived T cells. Disclosures Kufe: Canbas: Consultancy, Honoraria; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Hillstream BioPharma: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rosenblatt:Dava Oncology: Other: Education; Partner Tx: Other: Advisory Board; Parexel: Consultancy; Celgene: Research Funding; BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Imaging Endpoint: Consultancy. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: 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; Janssen: Consultancy.

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Vls-101 Is a Novel Therapeutic Antibody-Drug Conjugate (ADC) Targeting Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) in Richter's Syndrome (RS)

Blood ◽

10.1182/blood-2019-126827 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2856-2856 ◽

Cited By ~ 3

Author(s):

Tiziana Vaisitti ◽

Katti Jessen ◽

Thanh-Trang Vo ◽

Mira Ko ◽

Francesca Arruga ◽

...

Keyword(s):

Board Of Directors ◽

Tumor Cells ◽

Research Funding ◽

Cell Lymphoma ◽

Ex Vivo ◽

Normal Adult ◽

Advisory Committees ◽

High Affinity ◽

Pdx Models

ROR1 is a transmembrane receptor with tightly controlled expression during development. It is present on multiple tumor types but not on normal adult tissues. Hematological malignancies are often ROR1-positive, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and diffuse large B cell lymphoma (DLBCL). Given its unique pattern of expression, ROR1 represents a tumor-specific therapeutic target. The anti-ROR1 antibody, UC-961, is ahumanized IgG1 monoclonal antibody (mAb) that binds with high affinity to a specific extracellular epitope of human ROR1 receptor and can block Wnt5a-induced ROR1 signaling. Nonclinical studies document that UC-961 does not react with normal adult human tissues and selectively binds to tumor cells. Because of the antibody high specificity, rapid internalization, and trafficking to lysosomes, UC-961 appears ideally suited to serve as the targeting moiety for an anti-ROR1 ADC. Accordingly, we have developed VLS-101, a UC-961-linker-monomethyl auristatin E (MMAE) ADC that preserves the high-affinity binding and specificity of UC-961 and allows for ROR1-targeted intracellular release of MMAE. RS is an aggressive lymphoma, typically of DLBCL type, arising as transformation of CLL. Despite, progressive improvements in the therapy of CLL, very few effective treatment options exist for patients with RS. Using our recently established RS patient-derived xenografts (RS-PDXs), we explored the expression and signaling properties of ROR1 in RS and investigated the ex-vivo and in vivo effects of VLS-101. When assessed by flow cytometry (FACS), immunohistochemistry (IHC), and reverse-transcriptase-polymerase chain reaction (RT-PCR), 3 of 4 RS-PDXs showed ROR1 positivity (2 highly positive: 99% and 80% of cells; 1 medium/low positive: 25% of cells by FACS). The extent of ROR1 expression correlated among the 3 assays methods and was consistent with ROR1 expression data reported for non-RS DLBCL samples. When engaged by its ligand Wnt-5a, ROR1 activated downstream targets, Rac1 and RhoA, and induced phosphorylation of the p65 subunit of NF-kB and Jnk in RS cells. When cells purified from RS-PDX tumor masses were exposed to VLS-101 ex-vivo, the drug induced time- and dose-dependent apoptosis, as shown by increases in annexin V/propidium iodide and by Caspase-3 and PARP cleavage. VLS-101 efficacy was then assessed in vivo in both subcutaneous and systemic RS-PDX models. When palpable masses had formed in subcutaneous models, mice were randomly assigned to vehicle or VLS-101, bi-weekly treated for 3 consecutive weeks, and then compared for tumor growth and survival. In the 3 ROR1-expressing RS-PDX models, VLS-101 caused highly significant disease regressions. Complete regressions were observed even in RS-PDXs without universal ROR1 expression, suggesting a MMAE bystander effect. After treatment discontinuation, VLS-101-treated animals showed no tumor regrowth for several months and had significantly extended survival. Data were confirmed in systemic ROR1-expressing RS models in which tumor cells were intravenously injected and allowed to engraft for ~14 days before starting treatment. VLS-101 was administered with the same schedule adopted for the subcutaneous model. VLS-101 eliminated RS cells in bone marrow, peripheral blood, and spleen, increasing survival in VLS-101-treated animals relative to controls (approximately 20-50 days, depending on the RS-PDX model). Of note, no in vivo VLS-101 efficacy was observed in the ROR1-negative RS-PDX, confirming the specificity of VLS-101 targeting. VLS-101 showed no adverse effects on animal well-being or body weight. Collectively, these results indicate that ROR1 is expressed on RS cells where it can transduce pro-survival signals and can be diagnostically evaluated for selective targeting. Nonclinical data document that the MMAE-containing ADC, VLS-101, can cause RS-PDX apoptosis in vitro and can safely and selectively induce complete tumor regressions in in vivo models of RS derived from patient tumors with heavy prior clinical treatment and variable levels of ROR1 expression. Building on these types of results, a Phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with lymphoid cancers. Disclosures Vaisitti: VelosBio Inc.: Research Funding; Verastem Inc: Research Funding. Jessen:VelosBio Inc.: Employment. Vo:VelosBio Inc: Employment. Ko:VelosBio Inc: Employment. Allan:Sunesis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics LLC, an AbbVie company: Consultancy; Acerta Pharma: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Verastem Oncology, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Bayer: Consultancy. Furman:Acerta Pharma: Consultancy; Pharmacyclics: Consultancy; Beigene: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy; Incyte: Consultancy; Oncotracker: Consultancy; Verastem: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Janssen: Consultancy. Miller:VelosBio Inc.: Employment. Lanutti:VelosBio Inc.: Employment. Deaglio:iTeos Therapeutics: Research Funding; Verastem Inc: Research Funding; VelosBio Inc.: Research Funding. OffLabel Disclosure: The drug used in this project is an anti-ROR1-toxin-conjugated antibody

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L-Stereoisomer RNA Oligonucleotide Anti-SDF-1 (Nox-A12) Disrupts the Interaction of Multiple Myeloma Cells with the Bone Marrow Milieu In Vivo, Leading to Enhanced Sensitivity to Bortezomib

Blood ◽

10.1182/blood.v118.21.887.887 ◽

2011 ◽

Vol 118 (21) ◽

pp. 887-887

Author(s):

Aldo M Roccaro ◽

Antonio Sacco ◽

Phong Quang ◽

AbdelKareem Azab ◽

Patricia Maiso ◽

...

Keyword(s):

Bone Marrow ◽

Confocal Microscopy ◽

Board Of Directors ◽

Tumor Cells ◽

Research Funding ◽

Ex Vivo ◽

In Vivo Confocal Microscopy ◽

Advisory Committees

Abstract Abstract 887 Background. Stomal-cell-derived factor 1 (SDF-1) is known to be involved in bone marrow (BM) engrafment for malignant tumor cells, including CXCR4 expressing multiple myeloma (MM) cells. We hypothesized that de-adhesion of MM cells from the surrounding BM milieu through SDF-1 inhibition will enhance MM sensitivity to therapeutic agents. We therefore tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vivo and in vitro. Methods. Bone marrow (BM) co-localization of MM tumor cells with SDF-1 expressing BM niches has been tested in vivo by using immunoimaging and in vivo confocal microscopy. MM.1S/GFP+ cells and AlexaFluor633-conjugated anti-SDF-1 monoclonal antibody were used. Detection of mobilized MM-GFP+ cells ex vivo has been performed by flow cytometry. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence detection, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12 followed by bortezomib. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–200nM) treated primary MM BM stromal cells (BMSCs), in presence or absence of bortezomib (2.5–5nM), were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software (combination index: C.I. according to Chou-Talalay method). Results. We first showed that SDF-1 co-localizes in the same bone marrow niches of growth of MM tumor cells in vivo. NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BM stromal cells in vitro. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the PB of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 (50–100 nM) and bortezomib (2.5–5 nM) was observed (C.I.: all between 0.57 and 0.76). These findings were validated in vivo: tumor burden detected by BLI was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 followed by bortezomib was observed as compared to bortezomib alone treated mice (P <.05). Similarly, NOX-A12 + bortezomib combination induced significant inhibition of MM cell homing in vivo, as shown by in vivo confocal microscopy, as compared to bortezomib used as single agent. Conclusion. Our data demonstrate that the SDF-1 inhibiting Spiegelmer NOX-A12 disrupts the interaction of MM cells with the BM milieu both in vitro and in vivo, thus resulting in enhanced sensitivity to bortezomib. Disclosures: Roccaro: Roche:. Kruschinski:Noxxon Pharma AG: Employment. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Advisory Board, Research Funding.

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Targeting of JAK/STAT Signaling to Reverse Stroma-Induced Cytoprotection Against BCL2 Antagonist Venetoclax in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.32.32 ◽

2016 ◽

Vol 128 (22) ◽

pp. 32-32

Author(s):

Riikka Karjalainen ◽

Mihaela Popa ◽

Minxia Liu ◽

Mika Kontro ◽

Mireia Mayoral Safont ◽

...

Keyword(s):

Board Of Directors ◽

Stromal Cells ◽

Research Funding ◽

Stromal Cell ◽

Ex Vivo ◽

Intrinsic Resistance ◽

Advisory Committees ◽

Equity Ownership ◽

Refractory Aml

Abstract Several promising new, targeted agents are being developed for the treatment of AML. The BH3 mimetic venetoclax (ABT-199) is a specific inhibitor of BCL2, with results from a phase 2 study showing transient activity of venetoclax in relapsed/refractory AML (Konopleva et al, 2014). The bone marrow (BM) microenvironment is known to protect AML cells from drug therapy and we showed earlier that conditioned medium (CM) from BM stromal cells applied to AML patient cells conferred resistance to venetoclax, which could be reversed by the addition of the JAK1/2 inhibitor ruxolitinib (Karjalainen et al, 2015). Here, we investigated the mechanisms mediating the BM stromal cell induced resistance to venetoclax and its reversal by ruxolitinib. To identify the soluble factor(s) contributing to stroma-induced protection of BCL2 inhibition, we analyzed the cytokine content of 1) CM from the human BM stromal cell line HS-5, 2) CM from BM mesenchymal stromal cells (MSCs) isolated from AML patients, 3) supernatants from BM aspirates collected from AML patients, and 4) supernatants from BM aspirates collected from healthy donors. Although expression levels varied, the cytokines detected were similar among the different samples. In HS-5 CM, IL-6, IL-8 and MIP-3α were among the most abundant cytokines. In addition, gene expression analysis showed the receptors for these cytokines were expressed in AML patient samples. IL-6, IL-8 and MIP-3α were added individually to mononuclear cells collected from AML patients, which were then treated with venetoclax. However, none of the cytokines alone could mimic the reduced sensitivity to venetoclax conferred by the HS-5 CM suggesting that stromal cell induced cytoprotection is likely multi-factorial. Next we tested the effect of AML-derived BM MSCs on the ex vivo response of AML patient samples (n=8) to ruxolitinib or venetoclax alone or in combination in a co-culture setting. Apoptosis assays showed negligible effects of ruxolitinib at a concentration of 300 nM, while venetoclax at a dose of 100 nM induced reduction in the percentage of CD34+ AML cells. Co-treatment with venetoclax and ruxolitinib demonstrated synergistic effects in 6 out of 8 samples and significantly reduced the number of CD34+ AML cells. Mechanistic studies showed that ruxolitinib treatment inhibited the BM stromal medium-induced expression of BCL-XL mRNA on AML cells and the drugs in combination down-regulated BCL2, MCL1 and BCL-XL protein expression, which was in correlation with sensitivity to the drugs. To further evaluate the ability of the venetoclax and ruxolitinib combination to eradicate leukemic cells in vivo we used an orthotopic xenograft model of AML. NSG mice were injected with genetically engineered MOLM-13luc cells and after engraftment treated with venetoclax (25 mg/kg, i.p.), ruxolitinib (50 mg/kg BID, p.o) or both and imaged once per week for 4 weeks. At the end of the treatment period bioluminescent imaging showed significantly reduced leukemia burden in the ruxolitinib and venetoclax co-treated mice compared to controls demonstrating superior anti-tumor efficacy than either agent alone (Figure 1). In summary, our data demonstrate that the combined blockade of JAK/STAT and BCL2 pathways with ruxolitinib and ventoclax is synergistic in ex vivo co-culture models and in vivo in an AML mouse model. The addition of ruxolitinib was able to overcome intrinsic resistance to venetoclax by reducing expression of MCL1, a known escape mechanism of BCL2 inhibition. These results support further clinical investigation of this combination, particularly for relapsed/refractory AML. Disclosures Porkka: Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Wennerberg:Pfizer: Research Funding. Gjertsen:BerGenBio AS: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Heckman:Celgene: Research Funding; Pfizer: Research Funding.

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