Patient-derived Acute Myeloid Leukemia Models Research Articles

VIP943 Is a Novel CD123 Antibody Drug Conjugate with in Vitro and In Vivo Efficacy in Acute Myeloid Leukemia (AML) Models

Background: Current treatment options (cytarabine with anthracyclines) for patients with acute myeloid leukemia (AML) are often associated with severe and barely tolerable toxicities. In patients who tolerate induction therapy, the complete remission rate is 60%-80%; however, 50% of patients relapse due to the presence of leukemic stem cells (LSC). Antibody-drug conjugates (ADCs) are putatively designed to deliver potent drugs directly to cancer cells while sparing normal tissue; however, first-generation ADCs are associated with severe bone marrow and liver toxicity. VIP943 is a CD123-targeting ADC which uses a kinesin spindle protein inhibitor (KSPi) as a new payload class and a unique legumain linker designed to overcome liabilities of currently approved ADC therapies. The payload is delivered with a drug-antibody ratio of 6. It consists of the KSPi warhead which is modified with a CellTrapperTM moiety so it accumulates within the CD123-targeted tumor cell and cannot enter normal cells. Non-human primate toxicology studies demonstrated a highly favorable and differentiated safety profile for VIP943 (Kirchhoff et al, Cancers 2020). Here we characterize VIP943 in various patient-derived AML models. Methods: For co-localization experiments, MOLM-13 AML cell line was stained with labelled anti-CD123 antibody or isotype control and CytoPainter LysoGreen indicator reagent and analyzed by imaging flow cytometry. AML primary patient mononuclear cells from blood and bone marrow were treated with VIP943 for 24, 48, and 72 hours. Flow cytometry immunophenotyping for AML blasts and CD34+/CD38- LSCs was used to analyze these populations. An in vivo patient-derived AML PDX mouse model was treated with VIP943 (5 mg/kg IV every 7 days) or in combination with 5-azacytidine (2.5 mg/kg SC days 1-5 x 3) and venetoclax (50 mg/kg PO days 1-5 x 3). Cytokine release syndrome was analyzed comparing VIP943 to isotype control ADC, anti-CD123 antibody, and positive comparator antibodies by in vitro cytokine release assays (CRA) in two different assay formats using whole blood and isolated mononuclear cells from 10 healthy donors. Flow cytometric lymphocyte and leukemic immunophenotyping was performed from blood of non-human primates. A leukemia cell line panel drug screen with VIP943 was evaluated and baseline genomic features were correlated with response. Results: Co-localization studies confirm specific internalization and efficient intracellular trafficking to the lysosome of VIP943. In vitro treatment with VIP943 (66 nM, continuous exposure) delivers blast reductions of 37% after a 24h incubation with VIP943, 45% after 48h incubation and 83% after an 72h incubation. In the CD34+/CD38- cell population, a reduction of 71% was achieved. In a patient-derived AML model, VIP943 with 5-azacytidine and venetoclax led to complete tumor growth inhibition after 21 days showing superiority of this triplet therapy (complete regression: 8/9 mice) with high tolerability (maximum body weight loss -4.8 %). The immunotoxicological evaluation in both CRA formats and with the tested antibody concentrations (100µg, 10µg, 1µg) exerts no induction of cytokines by VIP943 compared to controls. Immunophenotyping demonstrates no impact of VIP943 treatment on lymphocytic subpopulations, whereas the CD123-positive basophils are reduced after 24h followed by an almost complete recovery. VIP943 IC50 ranges from 0.1nM-300nM in a leukemia cell line panel screen and features associated with sensitivity are investigated. Conclusions: VIP943 is a next-generation ADC with a differentiated safety profile from currently approved ADCs, including lack of in vitro cytokine release. In vitro and in vivo studies using patient-derived AML cells show VIP943 has favorable monotherapy and combination efficacy including targeting of leukemic stem cells, which drive relapse. These findings warrant evaluating VIP943 in clinical trials.

Abstract 6417: LY3410738, a novel inhibitor of mutant IDH1 is more effective than Ivosidenib and potentiates antileukemic activity of standard chemotherapy in preclinical models of acute myeloid leukemia (AML)

Abstract Acute myeloid leukemia is associated with the abnormal proliferation of myeloid progenitor cells unable to differentiate. Somatic gain-of-function mutations in isocitrate dehydrogenase (IDH) 1 occur in 10% of newly diagnosed AML patients. IDH1 mutations cause intracellular accumulation of the oncometabolite, 2-hydroxyglutarate (2-HG), which results in a hyper-methylation phenotype and a block in differentiation. Inhibitors of IDH1 mutant enzyme reduce levels of 2-HG, which relieves the differentiation block allowing AML cells to achieve terminal maturation. Recently, Ivosidenib, an IDH1 inhibitor, has been approved for use in AML patients. However, based on clinical findings, a fraction of the IDH1 mutant AML patients treated with Ivosidenib are primary refractory or relapse while on therapy. This raises the need for development of more potent inhibitors targeting IDH1. Lilly Research Laboratories have developed a potent covalent inhibitor of mutant IDH1, LY3410738 that modifies a single cysteine (Cys269) in an allosteric binding pocket and rapidly inactivates the enzyme, selectively inhibiting 2-HG production without affecting alpha-ketoglutarate (a-KG) levels. Here, we have assessed the activity of LY3410738 in IDH1 mutated patient-derived AML models and AML cell lines engineered to express wild-type IDH1 or mutant IDH1R132H. In vitro, LY3410738 displayed greater potency for inhibition of 2-HG production and differentiation of the IDH1 mutant cells compared to AG-120. Similarly, in vivo, we observed sustained 2-HG inhibition leading to a more robust and durable efficacy for LY3410738 with respect to AG-120. We next evaluated the combination activity of LY3410738 with Cytarabine and Azacitidine or the FLT3 inhibitor Midostaurin, the latter in FLT3-mutated AML. Combining LY3410738 with the chemotherapeutics resulted in increased efficacy, exhibiting a potent anti-leukemic effect, reduction of 2-HG level, and enhanced differentiation of the leukemic blasts in the mice. In addition, since IDH1 mutant AML cells have been shown to strongly depend on the anti-apoptotic Bcl-2 for the survival, we also combined LY3410738 with FDA approved Bcl-2 inhibitor, venetoclax. In vitro, isogenic cells with IDH1R132H mutation were more sensitive to the combination than wild-type IDH1-expressing cells. Importantly, the combination of LY3410738 with Venetoclax was also efficacious in an AML xenograft model derived from a patient refractory to AG-120. In conclusion, LY3410738 exhibits enhanced efficacy in IDH1 mutant AML PDX models in combination with Cytarabine, Azacitidine, Midostaurin and Venetoclax and demonstrates improved potency and durability compared to Ivosidenib. Citation Format: Vivian Salama, Nathan Brooks, Anna Skwarska, Lisa Kays, Paul Milligan, Katherine Newell, Kenneth Roth, Sandaruwan Geeganage, Raymond Gilmour, Steven M. Chan, Jean-Emmanuel Sarry, Mary Sabatier, Courtney DiNardo, Marina Konopleva. LY3410738, a novel inhibitor of mutant IDH1 is more effective than Ivosidenib and potentiates antileukemic activity of standard chemotherapy in preclinical models of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6417.

Identification of Novel Combination Therapies Active in BCL2 Inhibitor-Resistant Patient-Derived AML Models

Acute myeloid leukemia (AML) is an aggressive, heterogeneous malignancy. AML patients whose disease relapses on chemotherapy or are unfit for aggressive induction regimens have limited therapeutic options. Many patients benefit from the combination of venetoclax (BCL2i) and a hypomethylating agent (HMA) but this regimen is rarely curative. The addition of novel agents could provide improved benefit for relapsed/refractory patients. To identify such regimens, we screened a panel of 10 AML cell lines with combinations of venetoclax and novel targeted agents. The agents used spanned multiple mechanisms of action (e.g. DNA damage response, kinase signaling, pro-apoptotic agents) and are all in early clinical development. Cells were treated for 72hrs and viability was assessed by CellTiter-Glo. In several of the cell lines that were insensitive or partially sensitive to venetoclax (OCI-AML3, KG1a, MonoMac6, THP1), combinations with inhibitors of MCL1 (AZD5991), AURKB (AZD2811), and BRD4 (AZD5153) showed synergistic activity (Loewe synergy score >5, growth inhibition > 180%) (Table 1). We next asked if these combinations were active in patient-derived xenograft (PDX) models of AML. We established an ex vivo co-culture assay using the HS-5 bone marrow stromal cell line. AML PDX cells were isolated from mouse spleens and plated in 96-well format in direct co-culture with HS-5 cells or in HS-5-derived conditioned media. Cells were treated with three doses of each monotherapy and three doses of fixed ratio combination. Replicate screens using cells from individual mice on different days confirmed data were reproducible (r2=0.687) across animals engrafted with the same PDX. Drug response was similar between conditioned media and direct co-culture assays (r2=0.81). Venetoclax sensitivity varied across PDX models ex vivo. Notably, 2/5 PDX models screened (DFAM-68555 and DFAM-10360) were insensitive to both venetoclax and the combination of venetoclax + 5-azacytidine (HMA) ex vivo. Both models were established from untreated/1L patients and harbor TP53 mutations. Combination treatments did not add additional benefit over venetoclax monotherapy in the DFAM-10360 model. However, in DFAM-68555, AZD5153, AZD5991, and AZD2811 showed improved activity over venetoclax alone (67%, 54%, and 67% vs. 26% decrease in viability for venetoclax alone, respectively). Since combination strategies will likely be most impactful in patients refractory to or relapsed after venetoclax, we chose this venetoclax insensitive model to prioritize in vivo. To confirm the translatability of these findings, we designed a pilot in vivo study using DFAM-68555. Mice were randomized to receive vehicle, venetoclax + HMA, or venetoclax + AZD5153 when peripheral blood disease reached ~5% (hCD45+hCD33+ cells by flow cytometry). After two weeks of dosing, animals were sacrificed to evaluate disease burden in bone marrow (sternum), spleen, and peripheral blood. The model remained insensitive to venetoclax + HMA in vivo. The combination of AZD5153 with venetoclax decreased disease burden in blood and spleen compared to vehicle (30% and 42% hCD45+CD33+ cells by flow cytometry vs 70% and 95%, respectively) with similar efficacy seen by immunohistochemistry in the bone. Finally, we screened these venetoclax combinations in additional aggressive AML PDX models which were resistant or only partially responsive to venetoclax in vivo. Addition of AZD2811NP and AZD5991 to venetoclax was more effective than venetoclax alone and venetoclax + HMA in the bone marrow. The most active combination varied from model to model. Efficacy screening in additional models is ongoing to further build ex vivo to in vivo translation and prioritize development of specific combinations. Also ongoing is genomic and transcriptomic profiling of these PDXs to identify potential predictive biomarkers of combination activity. In summary, we developed an ex vivo screening platform to test clinically actionable combinations for activity in clinically relevant models. Using this platform and subsequent in vivo efficacy, we identified venetoclax combinations across multiple mechanisms (pro-apoptotic, cell cycle regulation, transcriptional regulation, DNA damage response) with activity in venetoclax-insensitive models. These results suggest potential therapeutic options to explore clinically for AML patients. Disclosures Andersen: AstraZeneca: Employment. Christie:AstraZeneca: Employment. Rosen:Astrazeneca: Employment. Maratea:AstraZeneca: Employment. Hattersley:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Cidado:AstraZeneca: Employment. Pulukuri:AstraZeneca: Employment. Saeh:AstraZeneca: Employment. Clark:AstraZeneca: Employment, Equity Ownership. Reimer:AstraZeneca: Employment. Mettetal:AstraZeneca: Employment.

A stemness screen reveals C3orf54/INKA1 as a promoter of human leukemia stem cell latency

AbstractThere is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

Single Doses of Antibody Drug Conjugates (ADCs) Targeted to CD117 or CD45 Have Potent In Vivo Anti-Leukemia Activity and Survival Benefit in Patient Derived AML Models

Single Doses of Antibody Drug Conjugates (ADCs) Targeted to CD117 or CD45 Have Potent In Vivo Anti-Leukemia Activity and Survival Benefit in Patient Derived AML Models

Single Doses of Antibody Drug Conjugates (ADCs) Targeted to CD117 or CD45 Have Potent In Vivo Anti-Leukemia Activity and Survival Benefit in Patient Derived AML Models

Allogeneic bone marrow transplant (BMT) is a potentially curative approach in patients with refractory or high-risk hematologic malignancies, like acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this procedure due to regimen-related mortality and morbidities. As a result, many eligible patients do not consider transplant and only 2/3 those transplanted are able to tolerate a reduced intensity conditioning regimen, which is associated with increased relapse (Scott, J Clin Onc 2017). Thus, safer and more effective conditioning agents with improved disease control are urgently needed. We developed two novel antibody drug conjugates (ADCs) conjugated to amanitin (AM) targeting CD117 (C-KIT, Pearse 2018), which is expressed on hematopoietic stem, progenitor, AML and MDS cells in >60% of patients (Ludwig, Haematologica 1997). CD45 (Palchaudhuri 2018) is expressed on all lympho-hematopoietic cells and hematologic malignancies except multiple myeloma. We aim to design ADC-based conditioning agents with the dual benefit of specifically depleting primary human hematopoietic stem progenitor cells (HSPCs) to enable BMT while reducing disease burden in leukemia models. ADCs were tested in xenograft murine models inoculated with human AML cells from immortalized cell lines (Kasumi-1, CD117 expressing leukemia; REH-Luc, CD45 expressing AML), and three AML patient-derived xenografts (PDX) expressing both CD117 and CD45 (2 of 3 are from patients that relapsed post allogeneic BMT). In the Kasumi model, a single dose of 0.3 mg/kg anti-CD117-AM on day 7 or 42 after inoculation markedly increased survival (median >240 days) compared to vehicle (median 76 days) or unconjugated anti-CD117 antibody (median 86.5 days) (n=6-8/group, p Anti-CD117-AM and anti-CD45-AM are potent anti-leukemia agents based on these data in humanized murine models with established AML. Together with prior reports on the potency of anti-CD117-AM and anti-CD45-AM as conditioning agents, these non-genotoxic ADCs may be useful in reducing disease burden in patients and in recipients of reduced-dose conditioning who are at high risk of disease relapse.

Single Doses of Antibody Drug Conjugates (ADCs) Targeted to CD117 or CD45 Have Potent In Vivo Anti-Leukemia Activity and Survival Benefit in Patient Derived AML Models

Background. Allogeneic bone marrow transplant (BMT) is a potentially curative approach in patients with refractory or high risk hematologic malignancies, such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Prior to transplant, patients are prepared with non-specific, high dose chemotherapy alone or in combination with total body irradiation, which are associated with early and late morbidities, including organ toxicities, infertility, secondary malignancies, and substantial risk of mortality. As a result, many eligible patients do not consider transplant and of those transplanted, 2/3 can only tolerate reduced intensity conditioning, which is associated with increased relapse rates (Scott et al. Journal of Clinical Oncology 2017, 1154-1161). Thus, safer and more effective conditioning agents with improved disease control are urgently needed. To meet this need, we developed two novel antibody drug conjugates (ADCs) conjugated to amanitin (AM) targeting CD117 (C-KIT, Pearse 2018), which is expressed on hematopoietic stem and progenitor cells and AML and MDS cells in >60% of patients (Ludwig et al. Haematologica 1997, 617-621), and CD45 (Palchaudhuri 2018) which is expressed on all lympho-hematopoietic cells and nearly all hematologic malignancies except multiple myeloma. The aim of the project was to design an agent with the dual benefit of depleting primary human hematopoietic stem progenitor cells (HSPCs) while reducing disease burden in leukemia models.Methods. ADCs were tested in xenograft murine models inoculated with human AML cells from immortalized cell lines (Kasumi-1, a CD117 expressing leukemia cell line, and REH-Luc, a CD45 expressing AML cell line tagged with luciferase), and three patient-derived xenografts (PDX) developed from FLT-3+NPM1+ AML samples (J000106132 [prior treatment with Allogeneic BMT, Sorafenib, Hydroxyurea, and Decitabine], J000106565 [M4/5, prior treatment with Induction chemotherapy, Consolidation HiDAC, Allogeneic BMT], J000106134 [M4, no prior treatment reported]) with varying growth kinetics (median survival of vehicle treated groups was 43, 63, 82 days post inoculation) that express both CD117 and CD45 (Jackson Laboratories).Results. In the Kasumi model, a single injection of 0.3 mg/kg anti-CD117-AM administered on day 7 or 42 after AML inoculation resulted in a marked increase in survival (median >240 days) compared to vehicle treated controls (median 76 days) or unconjugated anti-CD117 antibody (median 86.5 days) (n=6-8 mice/group, p<0.0001). In the REH-Luc model, a single injection of 1 mg/kg anti-CD45-AM on day 5 after AML inoculation resulted in longer survival by a median of 15 days compared to vehicle treated controls or unconjugated anti-CD45 antibody (n=10 mice/group, p<0.0001). For the three PDX, a single intravenous dose of ADCs (anti-CD117-AM, anti-CD45-AM, isotype-AM (ISO-AM), unconjugated anti-CD117 antibody, or vehicle PBS) were administered to AML-PDX animals when 2-5% blasts were observed in the blood. With 4-5 mice/group/AML-PDX model, survival was significantly increased in recipients of 1 mg/kg anti-CD117-AM, and 1 mg/kg anti-CD45-AM as compared to vehicle controls (Figure 1 and Table 1).Conclusions. Anti-CD117-AM and anti-CD45-AM are potent anti-leukemia agents based on these data in humanized murine models with established AML. Together with prior reports on the potency of anti-CD117-AM and anti-CD45-AM as conditioning agents, these non-genotoxic ADCs may be useful to reduce disease burden in patients with active disease and in recipients of reduced dose conditioning who are at high risk of disease relapse. [Display omitted] DisclosuresProctor:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Gabros:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Aslanian:Magenta Therapeutics: Employment, Equity Ownership. Pearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Kallen:Magenta Therapeutics: Employment, Equity Ownership. Sarma:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Ladwig:Magenta Therapeutics: Employment, Equity Ownership. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McDonagh:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Abstract 1958: Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models

Abstract Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in a variety of cancers and a small molecule inhibitor of Smoothened (SMO), Vismodegib, has been approved for the treatment of basal cell carcinoma, a disease frequently driven by Hh pathway signaling due to pathway mutations. SMO dependent Hh signaling has also been implicated in models of myeloid leukemia, primarily CML, where genetic loss of SMO or pharmacological inhibition limits disease progression in part through targeting the leukemic stem cell (LSC). Outside of BCR-ABL driven leukemia the role of Hh signaling and impact of SMO inhibition on disease progression and the LSC remains unclear. To explore the role of Hedgehog pathway signaling and interrogate responses to SMO inhibition in AML, a panel of primary AML patient-derived models was utilized to examine responses to PF-04449913, an oral small molecule SMO inhibitor currently in early phase clinical trials targeting myeloid malignancies. AML patient samples were characterized for Hh pathway expression levels and screened for responses to PF-04449913. Ex vivo treatment of AML bone marrow cells showed PF-04449913 was capable of inhibiting Hh pathway activity, reducing expression of key LSC regulators and decreasing populations of cells expressing LSC markers. Use of AML xenotransplant models to assess in vivo responses to PF-04449913 as single agent and in combination with Cytarabine have shown potential for combination efficacy of the two agents in select models suggesting patient selection strategies may be critical for SMO inhibitor-based therapies in AML. Citation Format: Amy Jackson-Fisher, Pamela Whalen, Mark Elliott, Melissa McMahon, Enhong Chen, Xianxian Zheng, Mark Ozeck, Donghui Huang, Paul Lira, Joseph Lee, Cathy Zhang, Justine Lam, Mary Spilker, Shibing Deng, Patrick Lappin, Penny Venne, Cynthia Heinlein, Annelie Schairer, Karen McLachlan, Todd VanArsdale. Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1958. doi:10.1158/1538-7445.AM2014-1958