KinN Therapeutics AS Research Articles

Development of Novel Highly Specific FLT3 Targeted Inhibitors for Combination or Sequential Therapy of Acute Myeloid Leukemia

More than 25% of acute myeloid leukemia (AML) patients have an activating mutation of the fetal-liver tyrosine kinase 3 (FLT3) receptor. FLT3 mutations vary, can be multiple, are often unstable length mutations located in the receptor's juxtamembranous domain (internal tandem duplication (ITD) mutations), and represent the strongest single marker for relapse of AML. When developing novel inhibitors of FLT3, it should be taken into careful consideration that there is a high inter- and intra-patient variability in the length and sequence repeated within ITD mutations. Recently, the first FLT3 targeting kinase inhibitor midostaurin was approved for combination therapy in FLT3 mutated AML, and several other kinase inhibitors are at present also in preclinical development. These inhibitors share broad kinase specificity, in several cases co-inhibiting FLT3 with structurally related targets like c-Kit and AXL. These targets may add therapeutic effect as well as adverse effects. Highly specific kinase inhibitors against varying FLT3-ITDs are to our knowledge currently not in clinical development.We are developing a novel class of highly specific FLT3-ITD targeting kinase inhibitors based on a well-established chemical backbone previously used in well-tolerated anti-viral therapy. These inhibitors, the BiKin2 compounds, are specifically developed to have superior selectivity towards FLT3-ITD over closely related type III tyrosine kinases, minimizing the chances of adverse off-target effects. We are screening the compounds to identify analogues that target a wide variety of FLT3-ITD mutations, and that can be combined with other BiKin2 analogues and/or with other targeted therapies in order to avoid development of acquired resistance during therapy.By employing cell lines expressing wild type (wt) FLT3 and ITD mutations of varying lengths we are currently testing more than 40 analogues of the BiKin2 compounds in in vivo and in vitro settings. Among the leukemic cell lines being used are the FLT3-mutated cells MOLM-13 and MV4-11 and the FLT3-wt cells OCI-AML3. Additionally, we are using the BA/F3 cells as a model of FLT3-ITD oncogene addiction. The BA/F3 cells are stably transduced to express FLT3-ITD of different lengths cloned from an AML patient at diagnosis and relapse, in addition to specific point mutations affecting the kinase domain. These screens give us valuable information on how the BiKin2 compounds inhibit and affect ITDs of varying length and sequences. Thus far these novel compounds have shown promising results in ITD mutated cell lines, as shown in Table 1 and Figure 1A.We have piloted an in vivo toxicity test (MTD) and proof-of-principle-experiments with the first compound showing promising selectivity for FLT3-ITD, BCI-332. We found no dose-limiting toxicity at doses up to 10 mg/kg (Figure 1B), and the drug also showed significant inhibition of tumor growth in a subcutaneous model of FLT3-ITD expressing MV4-11 (ITD/null) cells (Figure 1C). The FLT3 selectivity of BCI-332 at 1 µM over 250 kinases in vitro is superior with 90% versus below 20% inhibition.In the clinic, patients often develop resistance to FLT3 inhibitors due to secondary mutations within the mutated gene as well as selection of resistant leukemic clones. Therefore, we used a high throughput in vitro drug sensitivity and resistance test (DSRT) approach to evaluate the efficacy of the model compound BCI-332 in a combination screen together with 527 clinically approved and investigational drugs. Drug combinations were tested in both FLT3-ITD expressing (MOLM-13 wt/ITD) and FLT3-wt expressing (OCI-AML3 wt/wt) cells, and we identified several potential drug candidates that showed additive effect in combination with BCI-332 in both conditions (Figure 2). Potential combination candidates need to be further validated in more AML cell lines and in primary cells from AML patients with defined ITD mutational status.In summary, we employed a novel approach to develop FLT3-ITD targeting drugs by screening inhibitors for selectivity against multiple different length ITD mutations, in an attempt to identify FLT3-ITD specific inhibitors with minimal off-target inhibition. Ultimately, our goal is to identify one or several drug candidate(s) to be tested in a clinical trial setting, in combination or in sequences combined with other targeted therapies. [Display omitted] DisclosuresAmiable:BCI Pharma: Employment. Popa:Kinn Therapeutics AS: Employment. Guillon:BCI Pharma: Employment. Heckman:Novartis: Research Funding; Orion Pharma: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; IMI2 project HARMONY: Research Funding. McCormack:Kinn Therapeutics AS: Other: Co-ownership. Surleraux:BCI Pharma: Employment, Equity Ownership.

The Transmembrane Protein CD37 Is Not a Promising Therapeutic Target in Acute Myeloid Leukaemia

Acute myeloid leukaemia is a disease of the haematopoietic stem and progenitor cells distinguished by an impairment of proliferation, propagation and differentiation of the haematopoietic lineage[1]. AML is a heterogenous disease, where patient stratification and personalised therapeutic strategies are of importance[2]. The transmembrane protein CD37 has been associated with regulation of cell to cell interaction and proliferation in both B and T cells. Mice knockout studies have suggested that CD37 is not crucial for B cell development, however, it is highly expressed in malignant and suboptimal conditions and thus represent a good target for B-cell malignancies[3]. Pereira et al. reported that their anti-CD37 antibody-drug conjugate was a potential therapeutic in AML[4]. Their study revealed anti-leukemic effect in primary AML material and cell lines bothin vivoandin vitro. An investigation of CD37 expression in AML patients is needed to understand the relevance of CD37 immunotherapy. To investigate the relevance of targeted therapy of CD37 the chimeric mAb NNV003 anti-CD37 antibody was evaluated in AML samples. Single-cell time-of-flight mass cytometry (CyTOF), an established tool to analyse the immune signalling in bone marrow and peripheral blood, was used to investigate CD37 expression in 59 AML patient samples. A panel was designed to categorise hierarchical landscape of AML and the leukemic stem cell population (LSCs). CyTOF identified CD37 expression exclusively in the B-cell population of our 59 patients, whereas the AML blast population was negative for CD37 expression. Flow cytometry, however; identified 5/59 patients with CD37 expression in the leukemic blast population. Although previous studies show that an anti-CD37 drug conjugate showed anti-leukemic effectin vivoandin vitro, our results do not support that it is a promising therapeutic biomarker. We have uncovered that malignant expression of CD37 is not a feature of the AML patients investigated. We do not consider CD37 to be a promising and good target for precision therapy of AML. Kayser, S. and M.J. Levis, Advances in targeted therapy for acute myeloid leukaemia. Br J Haematol, 2018. 180(4): p. 484-500.Patel, J.P., et al., Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med, 2012. 366(12): p. 1079-89.Payandeh, Z., et al., Anti-CD37 targeted immunotherapy of B-Cell malignancies. Biotechnol Lett, 2018. 40(11-12): p. 1459-1466.Pereira, D.S., et al., AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody-Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML. Mol Cancer Ther, 2015. 14(7): p. 1650-60. Disclosures Heyerdahl: Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Dahle:Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Gjertsen:Novartis:Consultancy;KinN Therapeutics AS:Current equity holder in private company;Pfizer Inc:Consultancy;BerGenBio AS:Consultancy, Research Funding;Alden Cancer Therapy AS:Current equity holder in private company.Mccormack:KinN Therapeutics AS:Current equity holder in private company.

The Combination of AXL Inhibitor Bemcentinib and Low Dose Cytarabine Is Well Tolerated and Efficacious in Elderly Relapsed AML Patients: Update from the Ongoing BGBC003 Phase II Trial (NCT02488408)

The Combination of AXL Inhibitor Bemcentinib and Low Dose Cytarabine Is Well Tolerated and Efficacious in Elderly Relapsed AML Patients: Update from the Ongoing BGBC003 Phase II Trial (NCT02488408)

Cross-Intolerance with Bosutinib after Prior Tyrosine Kinase Inhibitors in Patients with Chronic Phase Chronic Myeloid Leukemia: BYOND Phase 4 Study

Cross-Intolerance with Bosutinib after Prior Tyrosine Kinase Inhibitors in Patients with Chronic Phase Chronic Myeloid Leukemia: BYOND Phase 4 Study

Efficacy and Safety of Bosutinib By Charlson Comorbidity Index in Previously Treated Patients with Chronic Myeloid Leukemia: Results from the Phase 4 BYOND Study

Background: Bosutinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) resistant/intolerant to prior therapy and newly diagnosed Ph+ chronic phase (CP) CML. The efficacy and safety of bosutinib by baseline comorbidities in patients with CML resistant/intolerant to prior TKIs were investigated. Methods: The phase 4 BYOND trial (NCT02228382) is further examining efficacy and safety of bosutinib (starting dose 500 mg/day) in patients with CML resistant/intolerant to prior TKIs. Comorbidities were analyzed using the Charlson Comorbidity Index (CCI), a validated measure of the influence of relevant comorbid conditions on mortality. Scores were derived from baseline data and patients grouped by CCI score 2-3, 4-5, and ≥6. This analysis was based on ≥1 year of follow-up and included patients with Ph+ CP CML. Results: 156 patients with Ph+ CP CML (n=42 [26.9%], 48 [30.8%], and 66 [42.3%] for CCI 2-3, 4-5, and ≥6) received bosutinib. Median treatment duration was 28.9, 23.9, and 20.1 months for CCI 2-3, 4-5, and ≥6; median dose intensity was 366.7, 385.3, and 291.5 mg/day. Across CCI groups, a substantial proportion of patients attained/maintained cytogenetic and molecular responses (Table). Grade 3/4 treatment-emergent adverse events (TEAEs) rates were 64.3%, 72.9%, and 80.3% for CCI 2-3, 4-5, and ≥6. Emerging grade 3/4 TEAEs differed between groups; diarrhea and increased alanine aminotransferase (ALT) were more common for CCI 2-3 (21.4% and 23.8%, respectively) and 4-5 (18.8% and 16.7%) and pleural effusion more common for CCI ≥6 (12.1%). For CCI 2-3, 4-5, and ≥6, 19.0%, 20.8%, and 31.8% of patients discontinued treatment due to AEs and 2.4%, 2.1%, and 9.1% due to insufficient response. Most common AEs leading to discontinuation in the respective groups were increased ALT and aspartate aminotransferase (7.1% each), increased ALT (8.3%), and pleural effusion (3.0%). 10 deaths occurred (n=0 [0%], 1 [2.1%], and 9 [13.6%] for CCI 2-3, 4-5, and ≥6), 8 due to AEs (none bosutinib-related), 1 CML-related, and 1 of unknown cause. No on-treatment transformations to advanced CML occurred. Conclusions: Across CCI groups, a majority of patients with Ph+ CP CML achieved/maintained cytogenetic and molecular responses, and only 1 CML-related death was reported. Patients with CCI ≥6 showed a trend toward higher rates of TEAEs, discontinuations due to AEs, and death. Results demonstrate efficacy of bosutinib across CCI scores, including patients with important comorbidities. However, CCI stratification may enable identification of patients at higher risk of developing TEAEs who require more careful monitoring. Disclosures Gambacorti-Passerini: Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding. Brümmendorf:Janssen: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Merck: Consultancy; University Hospital of the RWTH Aachen: Employment; Ariad: Consultancy. Gjertsen:Astellas: Consultancy; The Norwegian Cancer Society: Research Funding; BerGenBio: Consultancy; ERA PerMed: Research Funding; ACTII AS: Equity Ownership; Daiichi Sankyo: Consultancy; Seattle Genetics: Consultancy; Haukeland University Hospital / University of Bergen: Employment; KinN Therapeutics AS: Equity Ownership; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Helse Vest Health Trust: Research Funding; Research Council of Norway: Research Funding; EU Horizon 2020: Research Funding; BerGenBio AS: Membership on an entity's Board of Directors or advisory committees. Abboud:Jazz Pharma: Speakers Bureau; Novartis: Other: Member on an entity's Board of Directors or advisory committees (Ended 12/30/2017), Research Funding; Agios: Other: Member on an entity's Board of Directors or advisory committees (Ended 12/30/2017); Tetraphase Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; NKarta: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Bayer: Consultancy, Honoraria. Rosti:BMS: Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Abruzzese:BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Leip:Pfizer: Employment, Equity Ownership. Viqueira:Pfizer Inc: Employment, Equity Ownership. García Gutiérrez:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Incyte: Honoraria, Research Funding. Giles:Epigene Therapeutics Inc: Consultancy, Other: leadership, stock/other ownership ; Novartis: Consultancy; Actuate Therapeutics Inc: Employment. Hochhaus:Pfizer: Research Funding; Novartis: Research Funding; BMS: Research Funding; Incyte: Research Funding; MSD: Research Funding.

Durable Responses Observed in Elderly AML Patients Unfit for Intensive Chemotherapy with First-in Class Selective AXL Inhibitor Bemcentinib (BGB324) in Combination with LDAC: Phase II Open-Label Study

Durable Responses Observed in Elderly AML Patients Unfit for Intensive Chemotherapy with First-in Class Selective AXL Inhibitor Bemcentinib (BGB324) in Combination with LDAC: Phase II Open-Label Study

Moxetumomab Pasudotox-Tdfk in Heavily Pretreated Patients with Relapsed/Refractory Hairy Cell Leukemia (HCL): Long-Term Follow-up from the Pivotal Phase 3 Trial

Moxetumomab Pasudotox-Tdfk in Heavily Pretreated Patients with Relapsed/Refractory Hairy Cell Leukemia (HCL): Long-Term Follow-up from the Pivotal Phase 3 Trial

BGB324, an Orally Available Selective Axl Inhibitor Exerts Anti-Leukemic Activity in the First-in-Patient Trial BGBC003 and Induces Unique Changes in Biomarker Profiles

Introduction: The RTK Axl represents a therapeutic target promoting AML cell proliferation and survival by pleiotropic mechanisms. Signalling through Axl is also known to suppress immune reactions mainly by inhibiting pro-inflammatory responses of antigen presenting cells. BGB324 is an orally available selective potent inhibitor of Axl, which is currently being investigated in a Phase 1 a/b trial in AML and MDS. We report here the clinical data and the impact of BGB324 on the Axl signalling pathway. Furthermore, we evaluated the T cell receptor (TCR) repertoire in order to assess potential effects of BGB324 on the immune response in AML patients.Methods: Twenty-five patients (twenty-one with relapsed/refractory AML and four with MDS) were treated in a classical 3+3 dose escalation design. Three dose levels were explored 400/100 mg (loading dose d1-2/maintenance dose), 600/200 mg and 900/300 mg. Serial bone marrow samples (BM) were available from n=5 patients. By means of immunoblotting and ELISA we analyzed phosphorylation of Axl and its downstream targets Erk/MapK and Akt as well as the levels of soluble Axl (sAxl) and osteopontin in plasma. The TCRß repertoire was quantified by Next Generation Sequencing of DNA isolated from peripheral blood MNCs using an Illumina MiSeq sequencer. TCRß gene containing the entire Vß, Dß and Jß segments were amplified with BIOMED2-TCRß-A and -B primer pools. Using genomic DNA as template, the amplicons were tagged with Illumina adapters and indices in two consecutive PCR reactions. Demultiplexing and FastQ formated data output was generated by the MiSeq reporter. Analysis of TCRß data was performed on a Microsoft Cloud using our in-house analysis pipeline Pippa, which relies on MiXCR analysis tools.Results: Treatment was generally well-tolerated. Diarrhea and fatigue represented the most frequent adverse events, which were mostly CTC Grade 1 or 2. One DLT (QTc prolongation) occurred and the MTD has not yet been reached. Steady-state levels of BGB324 were reached between three and six days after initiation of treatment. One AML patient achieved a CRi of 5 months, a second achieved a PR and another achieved clearance of circulating blasts accompanied by peripheral blood count recovery > 3 months (ORR 10%). Four additional AML patients (25%) experienced disease stabilisation for more than four months. Two MDS patients experienced a PR.From the five patients in whom BM samples were available three experienced either an objective response or disease stabilization for more than four months and two had progressive disease. We could detect phosphorylated Axl (pAxl) before treatment and decreased pAxl level after 21 days of treatment in 3 out of 3 responders/patients with SD while could not detect pAxl before treatment in the patients with PD (n=5; p=0.025). The patients with response or SD showed inhibition of pErk and pAxl (2 out of 3) or no change of the activity of these Axl downstream pathways (1 out of 3). The patients with PD showed increased pErk and pAkt level after treatment (n=5; p=0.082).Treatment with BGB324 led to an increase in the levels of sAxl after 21 days of treatment in the plasma of 67% of analyzed patients (n=12). SAxl levels were positively correlated to compound exposure (n=12; r=0.829), indicating that sAxl might be used as a biomarker of target engagement. The highest level of sAxl was seen in the patient who achieved a PR. Osteopontin levels decreased in 67% of analyzed patients after three weeks of treatment with BGB324 indicating modulation of this protein by BGB324. However, there was no correlation between increases of sAxl and decreases of osteopontin in this patient cohort.Interestingly, we could demonstrate diversification of the TCR repertoire by higher abundance of small T cell clones after three weeks of therapy with BGB324 compared to pre-treatment levels in two out of four analysed patients.Conclusion: BGB324 is well tolerated in AML patients and exhibits anti-leukemic activity. BGB324 has on-target activity in AML patients and blocks the phosphorylation of Axl and its downstream pathways. Furthermore, BGB324 can induce a diversification of the TCR repertoire in AML patients and might hold potential as an immune-activating drug. DisclosuresSonja:BergenBio: Honoraria, Other: Travel Support, 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. Micklem:BerGenBio AS: Employment, Equity Ownership. Janning:Teva: Honoraria. Fiedler:Pfizer: Research Funding; GSO: Other: Travel; Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding; Kolltan: Research Funding; Ariad/Incyte: Consultancy; Novartis: Consultancy; Gilead: Other: Travel; Teva: Other: Travel. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

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

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. [Display omitted] DisclosuresPorkka: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.

JAK1/2 and BCL2 Inhibitors Synergize to Counter-Act Bone Marrow Stromal Cell-Induced Protection of AML

BACKGROUND AND OBJECTIVESThe bone marrow (BM) microenvironment supports the survival of leukemic cells and influences their response to therapeutic agents by promoting drug tolerance and resistance. Novel therapeutic strategies are therefore needed that can override the BM mediated protection of AML cells in patients undergoing drug treatment. To address this we used a high-throughput drug screening method to identify novel drug combinations to reverse stromal-induced cytoprotection against the BCL2 antagonist venetoclax in primary AML samples. METHODSSensitivity of mononuclear cells collected from 18 AML BM aspirates or peripheral blood samples to a range of BCL2 inhibitors and tyrosine kinase inhibitors (TKIs) was assessed either in mononuclear cell medium (MCM, Promocell) or in a 25% HS-5 stromal cell-conditioned medium plus 75% RPMI medium mix (CM) to mimic cytoprotective bone marrow conditions. Cell viability was measured after 72 h and dose response curves generated for each tested drug. Drug sensitivity scores were calculated based on the area under the dose response curve. For the drug combination studies single agents (venetoclax, WEHI-539, ruxolitinib) were added simultaneously at fixed concentrations to AML cells and incubated for 72 h either in the MCM or CM medium. Cell viability was measured using the CellTiter-Glo assay. The expression of BCL2 genes was measured by qPCR after incubating the AML patient cells in either MCM or CM for 48 h. RESULTSIncubation of primary AML cells in the CM culture condition led to reduced sensitivity to BCL2 family inhibitors, suggesting that stromal-derived factors in the CM promote cytoprotection. This effect was particularly pronounced for the selective BCL2 inhibitor venetoclax, where the CM-induced loss of sensitivity coincided with decreased BCL2 expression and increased BCL2L1 expression. In contrast, JAK inhibitors showed improved efficacy in CM compared to MCM culture conditions. To determine if the protective effects of CM stromal-like conditions against venetoclax could be diminished, the drug was tested in combination with the JAK1/2 inhibitor ruxolitinib using AML cells cultured in MCM or CM. When tested on AML cells from 4 patients with the FLT3-ITD alteration, we found that ruxolitinib rescued the sensitivity of venetoclax in leukemic cells in the presence of CM and the combination of two drugs exhibited synergistic effects in this setting. The combinatorial activity, however, was not recapitulated in the MCM condition. Since CM was found to induce BCL2L1 expression, venetoclax was also tested in combination with a BCLXLspecific inhibitor WEHI-539. Analogously to the ruxolitinib-venetoclax combination, synergistic activity between venetoclax and WEHI-539 was observed towards leukemic cells in the presence of CM. CONCLUSIONSBy applying a functional, drug-based approach to understand microenvironment-induced mechanisms of drug resistance in AML, we found that the activity of the selective BCL2 inhibitor venetoclax towards AML cells is adversely affected in stromal-based conditions, while JAK inhibitors, in contrast, exhibit increased efficacy in these conditions. Our results suggest stroma-derived cytokines induce JAK-STAT signaling in AML cells, which results in increased BCL2L1 expression and drives resistance to venetoclax. However, blocking JAK1/2 with ruxolitinib restores the sensitivity of AML cells to venetoclax. We found that JAK1/2 inhibitors such as ruxolitinib can act synergistically with BCL2/BCLXL inhibitors, suggesting clinically useful combination treatments. DisclosuresGjertsen: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. Porkka:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kallioniemi:Vysis-Abbot: Patents & Royalties; Medisapiens: Membership on an entity's Board of Directors or advisory committees; IMI-Project Predect: Research Funding; Roche: Research Funding; Pfizer: Research Funding. Wennerberg:Pfizer: Research Funding. Heckman:Celgene: Honoraria, Research Funding.

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

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. [Display omitted] DisclosuresGjertsen: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.

Elacytarabine Is More Effective Preclinically Than Cytarabine In Combination With Daunorubicin In Primary Patient Xenografts Of AML

Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis. The overall survival rate in AML patients has changed little in the last 20 years with overall survival rates for young patients (< 60 years) remaining at 35%. However, for elderly patients (> 60 years), survival is less than 10%. Despite the fact that AML represents one of the most molecularly characterized malignancies, the standard therapeutic option is unaltered in the last 15 years i.e. we still solely rely upon a combination of anthracycline and Cytarabine (Ara-C) as the cornerstone of induction therapy.Recently many potentially drugable targets have been discovered and while responses have been observed, these are generally amongst sub-sets of patients with good to intermediate prognostic factors and most often in combination with conventional therapy. Elacytarabine (ELA; CP-4055) is a fatty acid derivative (elaidic acid ester) of Ara- C, currently in undergoing phase II clinical trial as second-course remission-induction therapy in combination with anthracycline.In this study we determined the maximum tolerated dose for the combination of ELA with Daunorubicin (DAN) in immunodeficient NOD-scid-IL2Rγcnull (NSG) mice. Subsequently, we compared the preclinical efficacy of the combination of ELA (30 mg/kg; q.d.x5) or Ara-C (500 mg/kg; q.d.x2) with DAN (2.5 mg/kg; q.d.x3) in a primary patient xenograft model of refractory AML following relapse after Ara-C+DAN therapy (n = 24). Efficacy was evaluated employing a novel time-domain imaging strategy utilizing targeted monoclonal antibodies conjugated to near infrared dye that specifically recognize and bind to epitopes on AML cells enabling non-invasive monitoring of disease progression following therapy. Similarly, bone marrow aspirates were assayed for AML content by flow cytometry (CD45+/34+/33+) and IHC analysis.The combination of ELA and DAN was well tolerated in NSG mice xenografted with primary patient AML cells. While longitudinal near infrared optical imaging of AML disease dissemination illustrated lower disease burden in Ara-C + DAN treated mice than controls (p<0.05), those treated with ELA+DAN demonstrated significantly reduced disease progression, both in comparison to controls (p<0.001) and Ara-C+DAN (p<0.01) treated NSG mice. Flow cytometry analysis of bone marrow aspirates revealed similar reductions in ELA treated mice. Ultimately, combination of ELA+DAN exhibited significant increases in mean survival time (45 ± 4.6 days) over control (31.6 ± 1.7 days; p<0.0001) and Ara-C+DAN treated NSG mice (39 ± 3.2 days; p<0.039).ELA is more effective preclinically than Ara-C in combination with DAN in primary patient xenografts of refractory AML. ELA was found to be well tolerated and significantly increased survival in combination with Daunorubicin. Despite withdrawal of Elacytarabine as a single agent (following Phase III clinical trial earlier this year in relapsed and refractory AML versus investigators choice of treatment) our preclinical results demonstrate combined therapy of ELA+DAN may promise enhanced survival advantages over current standard therapy for AML patients. Disclosures:Popa:KinN Therapeutics AS: Employment. Gjertsen:KinN Therapeutics AS: Membership on an entity's Board of Directors or advisory committees. McCormack:KinN Therapeutics AS: Membership on an entity's Board of Directors or advisory committees.

Pharmacological Inhibition Of The SIRT1 Deacetylase With The Small Molecule Inhibitor Tenovin-6 Enhances Ablation Of FLT3-ITD+ LSC In Combination With TKI Treatment

Acute myeloid leukemia (AML) is propagated by small populations of leukemia stem cells (LSC). Internal tandem duplication (ITD) in the FMS-like tyrosine kinase-3 (FLT3) is the most frequent mutation seen in AML patients, and is associated with poor prognosis. However, FLT3 tyrosine kinase inhibitors (TKI) demonstrate only moderate clinical activity in FLT3-ITD AML patients. Persistent FLT3-ITD+ AML LSC may represent a source of relapse after treatment. Additional therapeutic strategies are required to target LSC and improve outcomes for FLT3-ITD+ AML patients. We have shown that the stress-related deacetylase SIRT1 is expressed at high levels in FLT3-ITD+ AML stem/progenitor cells and in cord blood CD34+ cells ectopically expressing FLT3-ITD. Inhibition of SIRT1 using RNAi or a potent small molecule inhibitor Tenovin-6 (TV-6) induces apoptosis in FLT3-ITD+ AML progenitors via p53-dependent mechanisms while sparing normal CD34+ cells suggesting a potential role for SIRT1 in FLT3-ITD+ AML LSC maintenance (Blood 2012,120: A1368). We now show that treatment of FLT3-ITD+ AML CD34+ cells with the TKI AC220 (20 nM) did not significantly reduce SIRT1 levels, increase p53 acetylation, or increase p53 target gene expression (p21, Bax, Puma, DR5, Noxa), suggesting that kinase independent mechanisms maintain SIRT1 expression and activity in TKI-treated cells. The combination of TV-6 (1μM) with AC220 (20nM) significantly reduced survival of primary human FLT3-ITD+ AML CD34+ cells compared with AC220 or TV-6 alone (combination 47±8% inhibition, AC220 alone 21±7%, TV-6 alone 25±5%, p=0.03, n=7), and significantly increased inhibition of AML CD34+ cell growth compared to AC220 and TV-6 alone (TV-6 44±7% inhibition; AC220 30±6%, TV+AC220 59±8%, p=0.04, n=7). TV-6 treatment increased p53 expression and activity in AC220-treated FLT3-ITD+ AML CD34+ cells, but did not affect FLT3 phosphorylation or STAT5 activation. Interestingly, TV-6 treatment also reduced survival of FLT3-ITD+ AML CD34+ cells expressing the D835Y mutation associated with TKI resistance (AC220 5±5% inhibition, TV 33±8%, p=0.04, n=3], suggesting SIRT1 inhibition can target TKI-resistant FLT3-ITD+ AML cells. We next tested the effect of TV-6, AC220 and the combination on primary cells from one untreated cytogenetically normal and FLT3-ITD+ patient engrafted in NSG mice (2×106 cells/mouse).12 weeks after transplantation, mice were treated with vehicle, AC220 (10mg/kg, gavage), TV-6 (100mg/kg, intraperitoneal), or the combination (n=7 per group) for 4 weeks. Treatment with TV reduced human cell engrafted in NSG mice (TV-6 2.9*107±1.2*107 human CD45+ cells/2 femurs vs. Control 5.3*107±2.6*107, after 4 weeks treatment, p=0.04). Importantly, the combination of TV and AC220 significantly reduced AML cell engraftment compared with AC220 alone (AC220 2.4*107±0.9*107 human CD45+ cells. Combination 1.2*107±0.6*107, p=0.01). Residual functional LSC were assessed by transplantation into secondary recipients. BM cells from the combination treatment group demonstrated significantly reduced engraftment of leukemia cells after secondary transplantation compared to AC220 treatment alone (16 weeks after secondary transplantation, AC220 1.8*106±0.6*106 human CD45+ cells/2 femurs, Combination 0.4*106±0.1*106, p=0.02, n=6). To determine the potential of this combination for FLT3-ITD+ AML patients refractory to standard chemotherapy, AML xenografts established using cells from a chemotherapy-resistant patient (n = 36) were treated with TV-6, AC220, or the combination vs. Standard of care (SOC, Ara-C + DNR), and vehicle control. Molecular imaging of primary patient xenografts with fluorescently labeled monoclonal antibodies after 4 weeks of therapy revealed significant differences between combination and single arm groups (vs. TV-6, p=0.0005, n=8; vs. AC220, p=0.003, n=7). Furthermore, TV-6 alone increased survival over controls (TV-6 vs. Control, p=0.02, n=6), and the combination demonstrated significantly improved survival over AC220 (p=0.02) and SOC (p=0.006. n=6). In conclusion, our studies indicate that elevated SIRT1 expression in FLT3-ITD AML cells is maintained after FLT3 TKI treatment. Inhibition of SIRT1 with TV-6 can enhance ablation of FLT3-ITD+ AML LSC in combination with FLT3 TKI treatment. Our results support further evaluation of inhibition of SIRT1 as a therapeutic strategy in FLT3-ITD+ AML. Disclosures:Popa:KinN Therapeutics AS: Employment.

Single Cell Analysis Of Protein Phosphorylation In Chronic Myeloid Leukemia Treated With Dasatinib, Eltrombopag, and Pegfilgrastim

IntroductionTyrosine kinase inhibitor (TKI) induced neutropenia and thrombocytopenia may be relieved with G-CSF and trombopoietin analogs. To characterize the signaling response in a CML patient treated with hematopoietic growth factors and TKI, we employed single cell analysis of phosphoproteins in serial samples. MethodsWe characterized the single cell phosphoprotein responses to treatment with dasatinib, eltrombopag, and pegfilgrastim in CML-like cell lines K562 and KCL-22, normal peripheral blood leukocytes and leukocytes from a patient starting on nilotinib. These data formed a basis for comparison with serial samples (Figure 1) from a CML patients after extensive wash-out before restarted on third-choice TKI, combined with eltrombopag and pegfilgrastim. For characterization of in vivo transformational potential CML patient bone marrow cells were engrafted in duplicate in NOG mice by femoral injection. Basal phosphorylation was determined using 17 phosphorylation specific antibodies: All staining panels contain the same 4 surface antibodies and a live-dead discriminator; CD33(P67.6) PerCP-Cy5.5, CD38(HB7) PE-Cy7, CD34(581) PE, CD45(MEM-28) PE-Dynamics590 and phospho c-PARP(Asp214) AlexaFlour-700. Two phosphospecific antibodies were added to each panel with the respective direct conjugated dye Alexa Flour 488 and Alexa Flour 647. The lowest median signal for each phosphoprotein in CML cells and lymphocytes, respectively, were used as reference value for calculation of basal phosphorylation. [Display omitted] ResultsA 56 y old male was diagnosed with CML (Sokal RR 0.75, Hasford RR 929, karyotype 46,XY,t(9;22)(q34;q11)[20], FISH 9q34 ABL/22q11.2 BCR dual fusion 62% of cells, PCR positive BCR-ABL e13e2/e14a2), developed prolonged thrombocytopenia and neutropenia on various dose levels of imatinib and nilotinib (neutrophile <1x109/L, <30x109/L) during the first 7 months after diagnosis. After a 6 months pause of TKI therapy: quantitative PCR BCR-ABL1/ABL1 25%IS, bone marrow biopsy with reduced normal megakaryocytes. Platelet supportive therapy with eltrombopag 50 mg daily was initiated 14 days before start with dasatinib 50 mg daily keeping platelet level above 50. After two more weeks pegfilgastim 6 mg was started, increasing neutrophils above 1.0x109/L.The CML bone marrow engrafted without symptomatic disease in NOG mice over 6 months monitoring, in contrast to engraftment with disseminated disease in an accelerated phase CML patient sample and the KCL-22 cell line. Phosphorylation of patient myeloid cells indicated STAT3(pY705) and STAT5(pY694) phosphorylation 2 and 3 hours after intake of eltrombopag. Dasatinib modestly inhibited SRC(pY418) and STAT3(pY705), but only transiently blunted the effect of eltrombopag. Pegfilgrastim strongly induced STAT3(pY705), STAT3(pS727), and SRC(pY418). Further analyses of cellular subsets and phospho-Abl will be presented. ConclusionEltrombopag kept stable levels of platelets on dasatinib treatment, and cell phosphorylation reflected persisting signaling induced by eltrombopag after seven days on combination therapy. Pegfilgrastim induced strong phosphorylation of STAT3(pY705) during dasatinib co-therapy. Phosphoprotein signal analysis of patient leukemic cells reflected the hematological response of dasatinib, eltrombopag, and pegfilgrastim and may be used to monitor therapy responses of and growth factors and targeted therapy.Phosphoprotein modulated early after start of treatment. (A) Each day 1-23 is analysed separately with its pretreatment sample as control and signal baseline. (B) Various treatment days are compared, using pretreatment control at day 1 used as baseline. (A) and (B) were analysed by flow cytometry at the same day, using bar code labelling, sample processing and analysis as previously described (Skavland et al. Blood Cancer J 2012). On day 16 dasatinib and eltrombopag is administrated concomitantly. Signalling responses in lymphocytes were distinct different from myeloid cells. Further determination of cellular subsets and their signalling responses will be presented. Disclosures:Popa:KinN Therapeutics AS: Employment.