Acute Lymphoblastic Leukemia Patient-derived Xenografts Research Articles

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19×anti-CD3 (blinatumomab) or anti-CD33×anti-CD3 bispecific antibodies mediate Tcell activation and direct Tcell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines invitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited invivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous Tcells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing Tcells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

Abstract 1905: The ROR1 antibody-drug conjugate, MK-2140, enhances the efficacy of established drugs in preclinical models of pediatric acute lymphoblastic leukemia

Abstract Introduction: Children diagnosed with acute lymphoblastic leukemia (ALL) experience ~90% likelihood of cure. However, the outcome for high-risk ALL and children who relapse remains poor. Receptor tyrosine kinase like orphan 1 receptor (ROR1) is a cell surface antigen primarily expressed in embryonic tissue, but also overexpressed in hematological malignancies. UC961 is a humanized IgG monoclonal antibody with high affinity for ROR1. MK-2140 (Zilovertamab vedotin/VLS-101) and VLS-211 are antibody-drug conjugates (ADCs) of UC961 with monomethyl auristatin-E or PNU payloads, respectively. This study evaluated the in vivo efficacy of MK-2140 and VLS211 against pediatric ALL patient derived xenografts (PDXs). Methods: PDXs were selected based on ROR1 mRNA (RNA-seq) and cell surface (flow cytometry) expression and grown as orthotopic disease in immune-deficient NSG mice. Engraftment and drug responses were assessed by flow cytometric enumeration of the proportion of human versus mouse CD45+ cells in peripheral blood (%huCD45+). Events were defined a priori as the %huCD45+ exceeding 25% or leukemia-related morbidity. MK-2140 (2.5 and 5.0 mg/kg) and VLS-211 (0.25 and 0.5 mg/kg) were administered intravenously, weekly x 3 weeks. Vincristine (0.15 mg/kg once weekly), dexamethasone (5 mg/kg daily x 5) and Lasparaginase (1,250 U/kg daily x 5) (VXL) were administered intraperitoneally for 3 weeks, followed by MK-2140 (5 mg/kg weekly x 3 weeks). Drug efficacy was assessed by event-free survival of treated (T) and control (C) groups and by stringent objective response criteria. In receptor occupancy/pharmacokinetic studies, a single dose of MK-2140 was administered and samples collected for analysis at discrete timepoints thereafter. Results: ROR1 mRNA and cell surface expression showed a significant correlation (R=0.83, P=0.021). MK-2140 significantly delayed disease progression in 4/7 PDXs compared to vehicle control (T-C, -2.6 to 20.0 days). At the highest dose, MK-2140 elicited objective responses (remissions) in two PDXs. VLS-211 significantly delayed disease progression in 5/7 PDXs (TC, -1.8 to 70.5 days), including 3 objective responses. Prior treatment with VXL followed by MK-2140 significantly delayed disease progression compared to VXL alone (T-C, 56.8 versus 35.2 days). MK-2140 bound to ROR1+ cells with near 100% occupancy within two hours of treatment, which was sustained for up to 96 h. Consistent with the receptor occupancy data, plasma levels of MK-2140 were maintained at >100 nM for at least 96 h after a single dose (5 mg/kg). Conclusions: MK-2140 and VLS-211 exerted significant single-agent in vivo activity against a panel of pediatric ALL PDXs. MK-2140 significantly enhanced the efficacy of an induction-type regimen and should be considered for further evaluation in a minimal residual disease setting. Citation Format: Richard B. Lock, Kathryn Evans, Ben Watts, Edward P. Bowman, Steven Neuhauser, Timothy Stearns, Jeffrey H. Chuang, Vivek M. Phillip, Katti A. Jessen, Emily L. Jocoy, Carol J. Bult, Beverly A. Teicher, Malcolm A. Smith. The ROR1 antibody-drug conjugate, MK-2140, enhances the efficacy of established drugs in preclinical models of pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1905.

Abstract C095: The menin inhibitor VTP-50469 enhances the in vivo efficacy of established drugs against preclinical models of aggressive infant MLL-r acute lymphoblastic leukemia

Abstract Introduction: Over 70% of infants with acute lymphoblastic leukemia (ALL) present with mixed lineage leukemia (MLL1/KMT2A) gene rearrangements (MLL-r ALL), which is associated with poor prognosis. MLL-r encodes oncogenic fusion proteins that cause aberrant chromatin histone methylation and leukemic transformation of hematopoietic progenitors. A characteristic of MLL-r leukemia is high expression of the FLT3 gene. Menin is critical for leukemic transformation by MLL fusion proteins. VTP-50469, the preclinical precursor of revumenib (SNDX-5613), is a potent and selective small molecule inhibitor of the Menin-MLL interaction that has shown impressive single-agent activity in preclinical models of MLL-r acute leukemia. Revumenib is currently undergoing clinical evaluation, therefore it was of interest to assess the in vivo activity of VTP-50469 in combination with established drugs against MLL-r ALL patient-derived xenografts (PDXs). Methods: VTP-50469 was tested as a single agent against a panel of 7 pediatric MLL-r ALL PDXs in immune-deficient (NSG) mice (120 mg/kg twice daily x 28 days via oral gavage). Combination studies used VTP-50469 formulated in chow (0.1%) available ad libitum for 28 days in combination with either an induction-type regimen of vincristine (0.15 mg/kg once weekly x 4 weeks), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off x 4 weeks, VXL) all administered intraperitoneally, or the FLT3 inhibitor gilteritinib (Xospata, 30 mg/kg x 28 days via oral gavage). Events were defined as >25% huCD45+ in the peripheral blood. Drug efficacy was assessed by event-free survival of treated (T) and control (C) groups by T-C, T/C measurements and stringent objective response criteria (progressive disease, PD; complete response, CR; maintained CR, MCR), and disease infiltration in the spleen and femoral bone marrow (BM) at 28 days post-treatment initiation. Results: Single-agent VTP-50469 elicited MCRs in 6/7 PDXs and significantly (P<0.0001) reduced leukemia infiltration of the spleen (7/7 PDXs) and BM (6/7 PDXs) compared to vehicle control mice. When administered in chow, VTP-50469 elicited a CR in the MLL-7 PDX (T-C 48.2 days, T/C 8.1) and a PD in MLL-8 (T-C 23.0 days, T/C 4.9). VTP-50469 combined with VXL elicited MCRs in both PDXs (MLL-7 T-C 77.0 days, T/C 12.3; MLL-8 T-C 49.1 days, T/C 9.3) and significantly delayed leukemia progression compared with VTP-50469 or VXL alone (P=0.0013). Moreover, VTP-50469 combined with VXL almost completely eradicated leukemia infiltration of spleens and BMs by both PDXs. VTP-50469 combined with gilteritinib did not result in therapeutic enhancement compared to each single agent arm in either PDX. Conclusions: The addition of VTP-50469 to a standard-of-care induction-type regimen (VXL) resulted in therapeutic enhancement against two infant MLL-r ALL PDXs, while VTP-50469 plus gilteritinib was substantially less effective. These results support further clinical evaluation of revumenib in combination with standard-of-care therapy in MLL-r leukemia. Citation Format: Richard B Lock, Kathryn Evans, Sophia Gawan-Taylor, Ben Watts, Timothy Stearns, Eric J Earley, Emily L Jocoy, Carol J Bult, Beverly A Teicher, Gerard M McGeehan, Malcolm A Smith. The menin inhibitor VTP-50469 enhances the in vivo efficacy of established drugs against preclinical models of aggressive infant MLL-r acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C095.

Abstract B134: Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate

Abstract Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk hematological malignancy with poor outcome, and new treatments are urgently required. We address this challenge by targeting cytokine receptor-like factor 2 (CRLF2), which is overexpressed in ~50% of Ph-like ALL cases conferring chemoresistance and survival of Ph-like ALL cells through JAK/STAT signaling. CRLF2 is an ideal target due to its role in leukemogenesis, limiting the likelihood of relapse associated with antigen loss, and its minimal expression in healthy tissue. Here, we developed a novel CRLF2-targeting antibody fragment-drug conjugate (CRLF2-FDC) and evaluated its efficacy in preclinical models. Methods: A single-chain anti-CRLF2 variable monoclonal antibody fragment (scFv-Fc) was engineered by conventional hybridoma technology, PCR and expression in HEK293T cells. The scFv-Fc was conjugated to the anti-microtubule drug DM1 via a non-cleavable linker. The selective association and internalization of the CRLF2-FDC was determined in vitrousing Ph-like ALL cell lines and their CRLF2-knockdown counterparts, and ex vivo using patient-derived xenograft (PDX) cells. The in vivo efficacy of the CRLF2-FDC (equivalent to 10 mg/kg scFv-Fc, 0.4 mg/kg DM1, once weekly x 4 weeks via intravenous injection) was tested against pediatric ALL PDX models in immune-deficient (NSG) mice, in comparison to scFv-Fc or DM1 alone. The efficacy of the CRLF2-FDC was further evaluated following two weeks of standard-of-care induction chemotherapy [vincristine (0.15 mg/kg, once weekly), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off (VXL)]. Treatment efficiency was calculated by subtracting the median event-free survival (EFS) of the control and treated groups (T-C) and stringent objective response criteria (progressive disease, PD; partial response, PR; complete response, CR; maintained CR, MCR) and leukemia infiltration in major organs at day 28 post treatment initiation. Results: CRLF2-dependent association (P≤0.01) and internalization of the CRLF2-FDC was confirmed both in vitro and in vivo in Ph-like ALL cells. While the scFv-Fc alone or free DM1 elicited PDs in a Ph-like ALL PDX in vivo (T-C 6 and 1.6 days, respectively, P=0.0013 for both), the CRLF2-FDC achieved a PR (T-C 32 days, P=0.0088). Besides targeted drug delivery, the CRLF2-FDC also demonstrated a significant antagonistic impact on STAT5 signaling in vivo 8 h after administration, suggesting a two-pronged therapeutic approach. Notably, the CRLF2-FDC achieved an MCR when administered after VXL (T-C 53.9 days, P <0.0001) compared to a CR (T-C 25.2 days, P <0.0001) by VXL alone. Furthermore, the CRLF2-FDC significantly decreased leukemic burden in major organs at 28 days post treatment initiation. Conclusions: This study describes the selective potency and promising preclinical activity of a novel CRLF2-FDC for the treatment of Ph-like ALL when administered either as a single agent or following standard-of-care induction therapy. Citation Format: Richard B Lock, Sara M.A. Mohamed, Peter Schofield, Karl-Heinz Friedrich, Daniel Christ, Maria Kavallaris, Narges Bayat. Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B134.

Abstract A016: Novel LEF1 gene structural variants in T-cell acute lymphoblastic leukemia Patient-Derived Xenografts models

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematological disorder characterized by uncontrolled proliferation of immature T-cell precursors. LEF1 (Lymphoid Enhancer-Binding Factor 1) is a transcription factor that plays a crucial role in the development of T-cells. Recent studies have shown that LEF1 can affect the expression of MYC, a proto-oncogene that is frequently dysregulated in cancer. The highly expressed LEF1 gene and potentially its variants (K86E and P106L) stimulate cell growth by upregulating the expression of MYC. In T-ALL, MYC is often overexpressed, the abnormal expression of MYC propels the advancement of T-ALL and sustains the function of leukemia stem cells. The interaction between LEF1 and MYC in T-ALL is complex and involves multiple regulatory pathways, particularly the Wnt/β-catenin signaling pathway. Several studies have revealed a significant correlation between the inactivation of LEF1 and the overexpression of MYC in T-ALL. Therefore, understanding the role of LEF1 in T-ALL pathogenesis may provide new insights into the development of novel therapeutic strategies for this aggressive disease. This study shows that the novel structural variants (SVs) found in the LEF1 gene could be associated with the decreased expression of MYC. Methods: Leukemia cells derived from 6 patients with T-ALL were inoculated intravenously into immunodeficient mice tails. The burden of tumor was monitored within the peripheral blood (hCD45+), and the leukemia cells were collected once human CD45 was above 80%. Optical Genome Mapping (OGM, Bionano Genomics) and Whole Genome Sequencing (WGS) techniques were used to detect SVs and mutations. The findings were subsequently validated using polymerase chain reaction (PCR). Results: Of the 6 T-ALL models that were analyzed, two of them had SVs in the LEF1 gene. Although the expression of the LEF1 gene did not show significant changes, the expression of MYC was observed to be downregulated. The OGM analysis revealed that the second and third exons of the LEF1 gene, which are located in the β-catenin binding region, were deleted at the DNA level. The lack of complete identity between the sequences at the breakpoints could be attributed to DNA repair mechanisms. At the RNA level, the two models had distinct LEF1 mutations - E60K and P106L. Conclusion: In two of the six T-ALL models, novel SVs were found in the LEF1 gene that may decrease MYC expression compared to control samples. Further research is required to understand the relationship between LEF1 deletion and the regulation of MYC expression, as well as the role of this phenomenon in the development of T-ALL. Targeting LEF1 has emerged as a potential therapeutic strategy for the treatment of T-ALL. Citation Format: Yueying Wang, Wubin Qian, Mengting Qin, Xiaobo Chen, Jia Xue, Sheng Guo. Novel LEF1 gene structural variants in T-cell acute lymphoblastic leukemia Patient-Derived Xenografts models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A016.

Identification of Mir-532-5p As a Reliable miRNA Reference in Acute Lymphoblastic Leukemia

In acute lymphoblastic leukemia (ALL) cure rates have reached >90%, however, therapy resistance and relapses occur. Hence, the major interest in leukemia research is the understanding of ALL biology and, based on that, the development of new targeted therapies. As patient-derived xenograft (PDX) mouse models, such as the NOD/SCID/human ALL xenotransplantation model, are systems resembling the human disease with leukemia engraftment and manifestation in bone marrow (BM), central nervous system (CNS), and spleen, they provide reliable models to study ALL biology and preclinically evaluate new treatment strategies. MicroRNAs (miRNAs) are described to post-transcriptionally regulate the expression of genes coding for critical regulators of leukemogenesis either functioning as tumor-suppressors or oncomirs. Moreover, ALL miRNA profiles can serve as biomarkers indicating disease stages and distribution and can be used for patient risk stratification. To reliably compare miRNA data across different samples and, most importantly, across various cohorts, data normalization with a constantly expressed miRNA reference in the respective samples is required. We aimed at identifying a reliable miRNA reference which shows a stable and constant expression in different ALL samples in a series of cell lines and PDX ALL specimens derived from different organ compartments. We analyzed the expression of 6 frequently used miRNA references (RNU6, RNU1A1, SNORD44, 5sRNA, miR-103a-3p and miR-532-5p) by real-time quantitative PCR. All together 9 ALL cell lines (NALM-6, REH, RS4;11, KOPN-8, UoCB6, RCH-ACV, MHH-CALL4, EU3, and HAL-01), 22 PDX specimens including CNS-, BM-, and spleen-derived ALL cells, and peripheral blood mononuclear cells of 6 healthy controls (HC-PBMCs) were investigated. Expression stabilities of the respective miRNA references were assessed applying four different algorithms (geNorm, BestKeeper, Normfinder, ∆CT). MiRNA scores were ranked according to their performance using RefFinder. Finally, the impact of using various miRNA references on data normalization was evaluated in different ALL and control samples. Analyzing the stability of miRNAs within the ALL cohort, we identified miR-532-5p in combination with miR-103a-3p (geNorm), miR-532-5p (BestKeeper and NormFinder), and miR-103a-3p followed by miR-532-5p (∆CT; mean standard deviation of miR-103a-3p and miR-532-5p: 2.215 and 2.220, respectively) as the most stably expressed miRNAs. Importantly, the RefFinder algorithm identified miR-532-5p as the most reliable reference. Next, we assessed the stability of miRNA expression in HC-PBMCs, and identified SNORD44 (NormFinder, BestKeeper, ∆CT) or SNORD44 in combination with RNU6 (geNorm) as the miRNAs showing the lowest variation. These data were further substantiated by using RefFinder which identified SNORD44 as the most reliable miRNA reference in HC-PBMCs. Interestingly, when we combined the cohorts of ALL and HC samples and applied RefFinder, miR-532-5p in combination with miR-103a-3p were identified as the most stable miRNAs. Last, we analyzed the influence of using expression levels of different miRNAs for data normalization. We assessed the expression of miR-181a-5p, a miRNA described to be frequently overexpressed in ALL cells as compared to HC-PBMCs and used expression data of different miRNAs for normalization: miR-532-5p (most stably expressed in ALL cohort), miR-532-5p/miR-103a-3p (combining the most stable miRNA references in ALL cohort and HCs), or SNORD44 (most stably expressed in HC-PBMCs). Interestingly and in line with its described upregulation in ALL, we identified a significant overexpression of miR-181a-5p in BM-derived PDX samples as compared to HC-PBMCs when using miR-532-5p or miR-532-5p/miR-103a-3p for data normalization. However, when SNORD44 was used as a reference, no differential expression of miR-181a-5p in HC-PBMCs and BM-ALL cells was found indicating a critical impact of the normalization reference. In summary, we identified miR-532-5p to be constantly expressed across different leukemia samples providing a reliable miRNA reference for data normalization in ALL miRNA expression studies. Moreover, these data clearly emphasize that the chosen reference critically depends on the cellular background of the specific samples and requires careful selection and verification within the respective sample types.

An antibody fragment-decorated liposomal conjugate targets Philadelphia-like acute lymphoblastic leukemia

Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is an aggressive B-ALL malignancy associated with high rates of relapse and inferior survival rate. While targeted treatments against the cell surface proteins CD22 or CD19 have been transformative in the treatment of refractory B-ALL, patients may relapse due to antigen loss, necessitating targeting alternative antigens. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in half of Ph-like ALL cases conferring chemoresistance and enhancement of leukemia cell survival. Therefore, targeting CRLF2 may reduce the likelihood of relapse associated with antigen loss. We developed a CRLF2-targeting single-chain variable fragment modified by the fragment crystallizable region (CRLF2 scFv-Fc) conjugated to a drug maytansinoid 1 (DM1)-DOPC liposomal conjugate, creating homogeneous CRLF2-targeted liposomes (CRLF2-DM1 LIP). Cellular association and internalization studies in a Ph-like ALL cell line, MHH-CALL-4, compared to its lentivirally transduced CRLF2-knockdown counterpart (KD-CALL-4) revealed excellent CRLF2-targeting efficiency of CRLF2-DM1 LIP. Moreover, CRLF2-DM1 LIP showed selective association and internalization ex vivo using Ph-like ALL patient-derived xenograft (PDX) cells with minimal reactivity with non-target cells. Cell apoptosis assays demonstrated the CRLF2-dependent potency of CRLF2-DM1 LIP in Ph-like ALL cell lines. This study is the first to highlight the therapeutic potential of a CRLF2-directed scFv-Fc-liposomal conjugate for targeting Ph-like ALL.

Th1 cytokines in pediatric acute lymphoblastic leukemia

Immune milieus play an important role in various types of cancer. The present study focuses on the effect of Th1 cytokines on pediatric acute lymphoblastic leukemia (ALL). The reaction of ALL cell lines and patient-derived xenografts (PDX) to the most important Th1 cytokines TNF-α (tumor necrosis factor alpha) and IFN-γ (interferon gamma) is analyzed and correlated with the respective cytokine receptors and the intracellular signaling molecules. ALL cell lines and ALL PDX display a great heterogeneity in cell death after incubation with TNF-α and IFN-γ. Several samples show a dose-dependent and additive induction of cell death by both cytokines; others do not react at all or even display an increased viability. Apoptosis is the main type of cell death induced by Th1 cytokines in ALL cells. Over all leukemia cells analyzed, IFN-γ receptor (IFNGR) shows a higher expression than both TNF-receptors, resulting in higher phosphorylation of STAT1 (signal transducer and activator of transcription) compared to phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) in the TNF pathway. The activation of STAT1 correlates with the amount of cell death after stimulation with Th1 cytokines. TNF-α and IFN-γ lead to heterogeneous reactions in ALL cell lines and ALL PDX but are able to induce cell death by apoptosis in the majority of ALL blasts. The correlation of a high expression of IFNGR and following activation of STAT1 with cell death indicates an important role for IFN-γ signaling in this setting.

In vivo activity of the dual SYK/FLT3 inhibitor TAK-659 against pediatric acute lymphoblastic leukemia xenografts.

While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains dismal. Spleen tyrosine kinase (SYK) is a prominent cytosolic nonreceptor tyrosine kinase in pediatric B-lineage ALL (B-ALL). Activating mutations or overexpression of Fms-related receptor tyrosine kinase 3 (FLT3) are associated with poor outcome in hematological malignancies. TAK-659 (mivavotinib) is a dual SYK/FLT3 reversible inhibitor, which has been clinically evaluated in several other hematological malignancies. Here, we investigate the in vivo efficacy of TAK-659 against pediatric ALL patient-derived xenografts (PDXs). SYK and FLT3mRNA expression was quantified by RNA-seq. PDX engraftment and drug responses in NSG mice were evaluated by enumerating the proportion of human CD45+ cells (%huCD45+ ) in the peripheral blood. TAK-659 was administered per oral at 60mg/kg daily for 21 days. Events were defined as %huCD45+ ≥ 25%. In addition, mice were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival and stringent objective response measures. FLT3 and SYK mRNA expression was significantly higher in B-lineage compared with T-lineage PDXs. TAK-659 was well tolerated and significantly prolonged the time to event in six out of eight PDXs tested. However, only one PDX achieved an objective response. The minimum mean %huCD45+ was significantly reduced in five out of eight PDXs in TAK-659-treated mice compared with vehicle controls. TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric ALL PDXs representative of diverse subtypes.

From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia

Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.

In vivo activity of the dual PI3Kδ and PI3Kγ inhibitor duvelisib against pediatric acute lymphoblastic leukemia xenografts.

Acute lymphoblastic leukemia (ALL) remains one of the most common causes of cancer-related mortality in children. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases, and aberrations in the PI3K pathway are associated with several hematological malignancies, including ALL. Duvelisib (Copiktra) is an orally available, small molecule dual inhibitor of PI3Kδ and PI3Kγ, that is Food and Drug Administration (FDA) approved for the treatment of relapsed/refractory chronic lymphocytic leukemia and small lymphocytic lymphoma. Here, we report the efficacy of duvelisib against a panel of pediatric ALL patient-derived xenografts (PDXs). Thirty PDXs were selected for a single mouse trial based on PI3Kδ (PIK3CD) and PI3Kγ (PIK3CG) expression and mutational status. PDXs were grown orthotopically in NSG (NOD.Cg-Prkdcscid IL2rgtm1Wjl /SzJAusb) mice, and engraftment was evaluated by enumerating the proportion of human versus mouse CD45+ cells (%huCD45+ ) in the peripheral blood. Treatment commenced when the %huCD45+ reached greater than or equal to 1%, and events were predefined as %huCD45+ greater than or equal to 25% or leukemia-related morbidity. Duvelisib was administered per oral (50mg/kg, twice daily for 28days). Drug efficacy was assessed by event-free survival and stringent objective response measures. PI3Kδ and PI3Kγ mRNA expression was significantly higher in B-lineage than T-lineage ALL PDXs (p-values <.0001). Duvelisib was well-tolerated and reduced leukemia cells in the peripheral blood in four PDXs, but with only one objective response. There was no obvious relationship between duvelisib efficacy and PI3Kδ or PI3Kγ expression or mutation status, nor was the in vivo response to duvelisib subtype dependent. Duvelisib demonstrated limited in vivo activity against ALL PDXs.

Abstract 2053: Structural variants detected by optical genome mapping in acute lymphoblastic leukemia patient-derived xenografts models

Abstract Background: The established classification of acute lymphoblastic leukemia (ALL) does not cover all high-risk patients because of the difficulty in detecting novel or rare structural variants (SVs). Furthermore, many SVs and formed gene fusions have adverse prognostic effects on hematological malignancies. This study aims to investigate SVs in 22 patient-derived xenografts (PDX) of ALL and to assess rare SVs and chromosomal aberrations, particularly hematological malignancy genes and gene fusions in different individuals detected by optical genome mapping (OGM). Methods: Leukemia cells from 16 patients with B-cell precursor acute lymphoblastic leukemia (B-ALL) and 6 patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into immuno-deficient mice, growth curves were monitored, and leukemia cells were collected after human CD45 was more than 80%. OGM (Bionano Genomics), RNA sequencing (RNA-seq) and whole genome sequencing (WGS) techniques were used to detect SVs and gene fusions. In validation, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) tests were performed to identify specific SVs. Results: On average, each model detected 42 rare SVs with known overlapping genes in different types by OGM: 12 insertions, 25 deletions, 1 inversion, 3 duplications, 2 inter-chromosomal translocations and 1 intra-chromosomal translocation. 5 rare SVs were found in multiple models (&amp;gt;=5), and two of them were never reported in B-ALL and T-ALL. Gene fusions were detected in all ALL models by OGM, and 17% were novel fusions. At the mRNA level, gene fusions were detected only in 19 models, and 24% of the predicted gene fusions by OGM were identical to the results of RNA-seq (high confidence results including pseudogenes). These two technologies could detect different fusion partners. For example, MLLT1 was seen at the mRNA level, and ACER1 was detected at the DNA level in the same KMT2A rearrangement model. Of the 16 B-ALL models, 6 were p190 BCR-ABL1 (a fusion of BCR exon 1 and ABL1 exon 2), 1 was p210 BCR-ABL1 (a fusion of BCR exon 13 and ABL1 exon 2) and 5 of 7 had IKZF1 gene deletions. 2 p190 BCR-ABL1 models were insensitive to imatinib and 2 p190 BCR-ABL1 models as well as the model with IKZF1 exons 4-7 deletion were sensitive to imatinib when treatment was initiated early in the disease. A novel ARL15-IKZF1 fusion was identified in one of the sensitive models. Deletion of the PRKAR2B gene was another rare SV in both models sensitive to imatinib besides the IKZF1 deletion. Conclusion: OGM assays were more comprehensive, and novel rare SVs were identified in ALL. Combining the results of gene fusions between different technologies will provide more accurate predictions for ALL classifications. The combination of aberrant SVs may synergize to influence the efficacy of imatinib, which warrants further investigations. Citation Format: Yueying Wang, Mengting Qin, JinPing Liu, Xiaobo Chen, Jia Xue, Sheng Guo. Structural variants detected by optical genome mapping in acute lymphoblastic leukemia patient-derived xenografts models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2053.

Niche-expressed Galectin-1 is involved in pre-B acute lymphoblastic leukemia relapse through pre-B cell receptor activation.

B-cell acute lymphoblastic leukemia (B-ALL) reflects the malignant counterpart of developing B cells in the bone marrow (BM). Despite tremendous progress in B-ALL treatment, the overall survival of adults at diagnosis and patients at all ages after relapse remains poor. Galectin-1 (GAL1) expressed by BM supportive niches delivers proliferation signals to normal pre-B cells through interaction with the pre-B cell receptor (pre-BCR). Here, we asked whether GAL1 gives non-cell autonomous signals to pre-BCR+ pre-B ALL, in addition to cell-autonomous signals linked to genetic alterations. In syngeneic and patient-derived xenograft (PDX) murine models, murine and human pre-B ALL development is influenced by GAL1 produced by BM niches through pre-BCR-dependent signals, similarly to normal pre-B cells. Furthermore, targeting pre-BCR signaling together with cell-autonomous oncogenic pathways in pre-B ALL PDX improved treatment response. Our results show that non-cell autonomous signals transmitted by BM niches represent promising targets to improve B-ALL patient survival.

Erratum: Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model.

An erratum was issued for:Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model. The Authors section was updated from: Claudia Manriquez Roman1,2,3,4,5 R. Leo Sakemura1,2 Fang Jin6 Roman H. Khadka6 Mohamad M. Adada1,2 Elizabeth L. Siegler1,2 Aaron J. Johnson6 Saad S. Kenderian1,2,6 1T Cell Engineering Laboratory, Mayo Clinic, Rochester, 2Division of Hematology, Mayo Clinic, Rochester, 3Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, 4Department of Molecular Medicine, Mayo Clinic, Rochester, 5Regenerative Sciences PhD Program, Mayo Clinic, Rochester, 6Department of Immunology, Mayo Clinic, Rochester to: Claudia Manriquez Roman1,2,3,4,5 R. Leo Sakemura1,2 Brooke L. Kimball1,2 Fang Jin6 Roman H. Khadka6 Mohamad M. Adada1,2 Elizabeth L. Siegler1,2 Aaron J. Johnson6 Saad S. Kenderian1,2,6 1T Cell Engineering Laboratory, Mayo Clinic, Rochester, 2Division of Hematology, Mayo Clinic, Rochester, 3Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, 4Department of Molecular Medicine, Mayo Clinic, Rochester, 5Regenerative Sciences PhD Program, Mayo Clinic, Rochester, 6Department of Immunology, Mayo Clinic, Rochester.

Venetoclax and dinaciclib elicit synergistic preclinical efficacy against hypodiploid acute lymphoblastic leukemia.

Hypodiploid acute lymphoblastic leukemia (ALL) is an aggressive blood cancer with a poor prognosis despite intensive chemotherapy or stem cell transplant. Children and adolescents with positive end-of-induction minimal residual disease have an overall survival lower than 30%. However, data regarding therapeutic alternatives for this disease is nearly nonexistent, emphasizing the critical need for new or adjunctive therapies that can improve outcomes. We previously reported on the therapeutic efficacy of venetoclax (ABT-199) in hypodiploid B-lineage ALL but with limitations as monotherapy. In this study, we set out to identify drugs enhancing the anti-leukemic effect of venetoclax in hypodiploid ALL. Using a highthroughput drug screen, we identified dinaciclib, a cyclin-dependent kinase inhibitor that worked synergistically with venetoclax to induce cell death in hypodiploid cell lines. This combination eradicated leukemic blasts within hypodiploid ALL patient-derived xenografts mice with low off-target toxicity. Our findings suggest that dual inhibition of BCL-2 (venetoclax) and CDK9/MCL-1 (dinaciclib) is a promising therapeutic approach in hypodiploid ALL, warranting further investigation to inform clinical trials in this high-risk patient population.

Hyperactive CREB subpopulations increase during therapy in pediatric B-lineage acute lymphoblastic leukemia.

Persistence of residual disease in acute lymphoblastic leukemia (ALL) during the initial stages of chemotherapy is associated with inferior survival. To better understand clonal evolution and mechanisms of chemoresistance, we used multiparameter mass cytometry, at single-cell resolution, to functionally characterize pediatric B-ALL cells at disease presentation and those persisting during induction therapy. Analysis of ALL cells from presentation samples (n=42) showed that the most abundant phosphosignals were pCREB, pH2AX and pHH3 and we identified JAK-STAT and RAS pathway activation in five of six patients with JAK or RAS genetic aberrations. The clonal composition of ALL was heterogeneous and dynamic during treatment but all viable cell clusters showed pCREB activation. Levels of pCREB in ALL cells were increased or maintained during therapy and high dimensional analysis revealed a subpopulation of ALL cells at presentation that was positive for pCREB/pHH3/pS6 which increased during treatment in some patients, implicating this signaling node in conferring a survival advantage to multi-agent induction therapy. The small molecule CREB inhibitor, 666-15, was shown to reduce CREB transcriptional activity and induce apoptosis in ALL patient-derived xenograft cells of varying cytogenetic subtypes in vitro, both in the presence and absence of stromal support. Together, these data suggest that the cAMP signaling pathway may provide an opportunity for minimal residual disease-directed therapy for many patients at high risk of relapse.

Pediatric Preclinical Testing Consortium Evaluation of the MCL1 Inhibitor, AMG 176, Against Xenograft Models of Acute Lymphoblastic Leukemia

Introduction: Children diagnosed with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, however the outcome for high-risk subtypes remains poor. Evasion of apoptosis is a hallmark of cancer, with dysregulation of the BCL-2 family of proteins often reported (Delbridge et al, Nat Rev Cancer 16:99-109, 2016). The BCL-2 family consists of the anti-apoptotic MCL1, BCL-2 and BCLXL as well as pro-apoptotic proteins. AMG 176, a first-in-class MCL1 inhibitor, previously demonstrated potent apoptosis-inducing activity in the nanomolar range against cell lines and patient explants of human hematological malignancies (Caenepeel et al, Cancer Discov 8:1582-97, 2018). Furthermore, discontinuous oral administration of AMG 176 inhibited the growth of human acute myeloid leukemia and multiple myeloma tumor xenografts in vivo at well-tolerated doses. AMG 176 has entered clinical evaluation for patients with hematological malignancies. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium (PPTC) to evaluate the in vivo efficacy of AMG 176 against its patient-derived xenograft (PDX) models of pediatric ALL. Methods:MCL1, BCL-2 and BCLXL mRNA expression was quantified by RNA-seq (http://pedcbioportal.kidsfirstdrc.org). For in vitro cytotoxicity assays, cells were treated with AMG 176 (10 μM - 1 pM) or DMSO control for 24 h and cell viability was determined by AlamarBlue assay and expressed as IC50values. AMG 176 was evaluated in vivo against a panel of 38 ALL PDXs in a single mouse trial format. Each PDX was inoculated via tail vein injection into one NSG mouse (1-5 x 106 cells per mouse). Engraftment and drug responses were determined by enumerating the proportion of human versus mouse CD45+ (%huCD45+) cells in the peripheral blood (PB) at weekly intervals. Treatment with AMG 176 (provided by Amgen, Inc.) began once the %huCD45+ was ≥1% in each mouse and consisted of 60 mg/kg via oral gavage 2 days on, 5 days off for 2 weeks. The experimental endpoint was pre-defined as the %huCD45+ reaching 25% in the PB. Drug efficacy was assessed by event-free survival (EFS) and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). For the pharmacokinetic (PK) study naïve NSG mice were treated with either vehicle or AMG 176 (60 mg/kg) and groups of 3 mice harvested up to 24 h post-treatment. Plasma samples were collected for analysis of AMG 176 using acetonitrile protein precipitation and LC-MS/MS with gradient elution on a Phenomenex Kinetex XB-C18 (3.0 x 50 mm, 2.6 µm) analytical column. Results:MCL1 mRNA expression ranged from 21-282 FPKM across a panel of 90 ALL PDXs representing 6 broad ALL subtypes (B-cell precursor, BCP-ALL; Ph+-ALL; Ph-like ALL; mixed-lineage leukemia-rearranged, MLLr-ALL; T-cell ALL, T-ALL; and early T-cell precursor ALL, ETP-ALL). Thirty eight of the 90 ALL PDXs were selected for in vivo evaluation of AMG 176 activity based on their profiles of MCL1, BCL-2 and BCLXL mRNA expression. AMG 176 was well tolerated in the efficacy study, with only 3 mice experiencing >10% weight loss. Mouse EFS values ranged from 3.5 to 47.8 days from treatment initiation, however no PDX achieved an objective response (remission) with all but one PDX exhibiting progressive disease. The Ph-like ALL PDX, ALL-102 (MCL1 expression 71.8 FPKM) scored a stable disease and had the highest EFS value (47.8 days). In an effort to explain the poor in vivo activity of AMG 176 several follow-up studies were performed. First, the in vitro IC50 values elicited by the AMG-176 stock against 5 leukemia cell lines (MOLM-13, 1.6 µM; NB-4, 2.0 µM; Kasumi-1, 1.6 µM; THP-1, 1.2 µM; and U-937,1.3 µM) were comparable to published data. Second, C18 LC-MS analysis revealed major and minor peaks consistent with theoretical peaks with negligible degradation. Third, PK study results demonstrated that the AMG 176 plasma concentration peaked at 8.5 µM, one hour after administration, with an AUC of 87.7 µM*hr and t1/2 of 4.1 h. This AUC was markedly lower than that in athymic nude mice treated at the same dose (1,120 µM*hr). Conclusions: AMG 176 exhibited modest in vivo activity against a large range of pediatric ALL PDXs, with the best response defined as stable disease in a single PDX. PK data suggest that AMG 176 systemic exposures in NSG mice may not be sufficient to elicit meaningful and prolonged regressions in pediatric ALL PDXs. (Supported by NCI Grants CA199000 and CA199222)

The IL7R-Antagonist OSE-127 Blocks Acute Lymphoblastic Leukemia Development Via a Dual Mode of Action

Acute lymphoblastic leukemia (ALL) may arise from precursor B or T cells (BCP- and T-ALL, respectively) and treatment is based on polychemotherapy. There is an urgent need for novel immunotherapy targets to reduce therapy toxicity and to target relapsed/refractory (r/r) disease, especially in T-ALL where immunotherapy approaches are scarce. The Interleukin-7-receptor (IL7R) has been shown to play a pivotal role in the development of both BCP-ALL and T-ALL (Geron et al. 2022; Silva et al. 2022). The IL7Rα-chain (CD127)-targeting antibody OSE-127 has a good safety profile in healthy volunteers (NCT03980080) and has entered phase 2 trials in Ulcerative Colitis (NCT04882007) and Sjögren's syndrome (NCT04605978). We previously reported high preclinical efficacy of OSE-127 in BCP-ALL patient derived xenograft (PDX) models of different cytogenetic backgrounds (Lenk and Baccelli et al. ASH, 2021). However, the preclinical efficacy of OSE-127 in T-ALL had not yet been investigated and the mechanism of action underlying its anti-leukemic efficacy remained unclear. First, we investigated the in vivo efficacy of OSE-127 treatment on BCP-ALL and T-ALL cells by conducting a preclinical phase 2-like PDX study. Two NSG mice per patient were injected with BCP-ALL (n=13) or T-ALL (n=9) PDX cells (including 7 r/r ALL samples), randomly assigned into treatment groups and OSE-127 therapy was initiated when 1% leukemic blasts were detected in the peripheral blood (PB). OSE-127 treated mice showed a reduction of PB blasts in 21/22 (95%) PDX samples in vivo when the corresponding control mice showed clinical signs of overt leukemia (Figure 1A). This translated into a 1.6-fold prolongation of median survival (MS) of OSE-127 treated PDX mice (P<0.0001, Figure 1B). Of note, blast reduction strongly correlated with CD127 expression levels in the samples (Pearson r=0.5372, P=0.0099). Next, we characterized the mode of action of OSE-127. As expected, OSE-127 blocked STAT5 phosphorylation in IL7-dependent ALL cell lines and PDX samples. As OSE-127 is not internalized by CD127 expressing cells (Belarif et al. 2018), we investigated whether OSE-127 might also exert immune-mediated effects. We found that OSE-127 did not promote antibody-dependent cellular cytotoxicity (ADCC), but efficiently induced macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) in BCP- and T-ALL cell lines and PDX cells in vitro, in a manner dependent on CD127 expression levels (Pearson r=0.5000, P=0.0352, Figure 1C-D). In line with these observations, inactivating mutations of the Fc fraction of OSE-127 (OSE-127-LALAPG) prevented ADCP of ALL cells in vitro, while conserving its antagonistic properties on IL7R signaling. To further evaluate the contribution of ADCP to the anti-leukemic activity of OSE-127, we compared the in vivo efficacy of OSE-127 and OSE-127-LALAPG in ALL samples with dysfunctional IL7R signaling (e. g., DND41 cells showing constitutive pathway activation due to IL7Rα mutations or NALM6 cells being absent of P-STAT5 induction upon IL7 stimulation). In this context, OSE-127-LALAPG did not impact leukemic engraftment as compared to control treatment, while OSE-127 significantly reduced leukemic burden, thereby substantiating that ADCP contributes to the anti-leukemic efficacy of OSE-127 (Figure 1E). Finally, we conducted a phase2-like PDX overt leukemia study testing OSE-127 alone or in combination with Dexamethasone/Vincristine/PEG-Asparaginase chemotherapy employing PDX samples with high CD127 expression (6 BCP-ALL and 3 T-ALL-PDX samples with >50% CD127+ cells in flow cytometry analysis). OSE-127 alone significantly prolonged the survival of PDX mice in comparison to control treatment (MS 106d vs 79d, P=0.0071), which was comparable to the effect of chemotherapy (MS 106d vs 79d, P=0.0037). Importantly, the strongest effects were observed when combining OSE-127 with chemotherapy (MS 79d vs undefined, P=0.0003, Figure 1F). Overall, we propose OSE-127 as a novel potential immunotherapeutic agent for the treatment of BCP-ALL and T-ALL, particularly in r/r disease and in combination with chemotherapy. Due to its dual mode of action comprising ADCP induction and IL7R-pathway blockade, OSE-127 could be efficacious in CD127+ ALL with and without IL7R pathway alterations. L.L. and I.B. contributed equally to this work. N.P. and D.M.S. share senior authorship Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The first Japanese biobank of patient-derived pediatric acute lymphoblastic leukemia xenograft models.

A lack of practical resources in Japan has limited preclinical discovery and testing of therapies for pediatric relapsed and refractory acute lymphoblastic leukemia (ALL), which has poor outcomes. Here, we established 57 patient-derived xenografts (PDXs) in NOD.Cg-Prkdcscid ll2rgtm1Sug /ShiJic (NOG) mice and created a biobank by preserving PDX cells including three extramedullary relapsed ALL PDXs. We demonstrated that our PDX mice and PDX cells mimicked the biological features of relapsed ALL and that PDX models reproduced treatment-mediated clonal selection. Our PDX biobank is a useful scientific resource for capturing drug sensitivity features of pediatric patients with ALL, providing an essential tool for the development of targeted therapies.

Targeting TPC2 sensitizes acute lymphoblastic leukemia cells to chemotherapeutics by impairing lysosomal function

Despite novel therapy regimens and extensive research, chemoresistance remains a challenge in leukemia treatment. Of note, recent studies revealed lysosomes as regulators of cell death and chemotherapy response, suggesting this organelle is a novel target for chemosensitization. Interestingly, drug-resistant VCR-R CEM acute lymphoblastic leukemia (ALL) cells have an increased expression of the lysosomal cation channel Two-Pore-Channel 2 (TPC2) compared to drug-naïve CCRF-CEM ALL cells. Concurrently, knockout (KO) of TPC2 sensitized drug-resistant VCR-R CEM cells to treatment with cytostatics. The chemosensitizing effect could be confirmed in several cell lines as well as in heterogeneous, patient-derived xenograft ALL cells, using the pharmacological TPC2 inhibitors naringenin and tetrandrine. We reveal that a dual mechanism of action mediates chemo sensitization by loss of lysosomal TPC2 function. First, because of increased lysosomal pH, lysosomal drug sequestration is impaired, leading to an increased nuclear accumulation of doxorubicin and hence increased DNA damage. Second, lysosomes of TPC2 KO cells are more prone to lysosomal damage as a result of morphological changes and dysregulation of proteins influencing lysosomal stability. This leads to induction of lysosomal cell death (LCD), evident by increased cathepsin B levels in the cytosol, truncation of pro-apoptotic Bid, as well as the reversibility of cell death by co-treatment with the cathepsin B inhibitor CA-074Me in TPC2 KO cells. In summary, this study establishes TPC2 as a novel, promising, druggable target for combination therapy approaches in ALL to overcome chemoresistance, which could be exploited in the clinic in the future. Additionally, it unravels LCD signaling as an important death-inducing component upon loss of TPC2 function.