No abstract

Activated interleukin-7 receptor signaling drives B-cell acute lymphoblastic leukemia in mice.

CD20-directed immunotherapies, such as rituximab, are mainstays of clinical management of mature B-cell neoplasms, previously known as non-Hodgkin lymphomas (NHL). More recently, the bispecific CD20/CD3 antibody mosunetuzumab was approved in the EU and US for treatment of relapsed or refractory follicular lymphoma (FL). The clinical efficacy of these CD20-directed therapies is limited by instances of intrinsic or acquired resistance, frequently mediated by complete or partial loss of CD20 expression. For example, in most chronic lymphocytic leukemia (CLL) at diagnosis and in a subset of post-mosunetuzumab FL relapses, CD20 protein levels are inexplicably low, despite near normal levels of CD20 mRNA and no detectable CD20 genetic variants. Here, we show that in both normal and malignant B-cells, CD20 down-regulation commonly occurs via alternative splicing in its 5′ UTR. This event generates an extended 5' UTR isoform (V1) with a stem-loop structure and upstream open reading frames (uORFs), which cooperatively inhibit translation. This translation-deficient V1 was abundant in most CLL, in a CD20-negative post-mosunetuzumab FL relapse, and in a subset of diffuse large B-cell lymphoma (DLBCL) where it correlated with low CD20 protein levels. In a panel of B-cell lines, knockout of the Sam68/KHDRBS1 splicing factor with multiple putative binding sites in V1 shifted CD20 splicing toward translation-competent variants and increased CD20 levels. Even more profound effects on CD20 expression were observed by modulating CD20 splicing with Morpholino oligomers, which led to enhanced rituximab-mediated cytotoxicity. Thus, this widespread splicing-mediated mechanism of CD20 antigen loss potentially could be targeted to enhance CD20-directed immunotherapies.

Chimeric antigen receptor (CAR) T cell immunotherapies targeting CD19 or CD22 induce remissions in the majority of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), although relapse due to target antigen loss or downregulation has emerged as a major clinical dilemma. Accordingly, great interest exists in developing CAR T cells directed against alternative leukemia cell surface antigens that may help to overcome immunotherapeutic resistance. The fms-like tyrosine kinase 3 receptor (FLT3) is constitutively activated via FLT3 mutation in acute myeloid leukemia (AML) or wild-type FLT3 overexpression in KMT2A (lysine-specific methyltransferase 2A)-rearranged B-acute lymphoblastic leukemia (ALL), which are associated with poor clinical outcomes in children and adults. We developed monovalent FLT3-targeted CAR T cells (FLT3CART) and bispecific CD19xFLT3CART and assessed their anti-leukemia activity in preclinical models of FLT3-mutant AML and KMT2Arearranged infant ALL. We report robust in vitro FLT3CART-induced cytokine production and cytotoxicity against AML and ALL cell lines with minimal cross-reactivity against normal hematopoietic and non-hematopoietic tissues. We also observed potent in vivo inhibition of leukemia proliferation in xenograft models of both FLT3-mutant AML and KMT2A-rearranged ALL, including a post-tisagenlecleucel ALL-to-AML lineage switch patient-derived xenograft model pairing. We further demonstrate significant in vitro and in vivo activity of bispecific CD19xFLT3CART against KMT2A-rearranged ALL and posit that this additional approach might also diminish potential antigen escape in these high-risk leukemias. Our preclinical data credential FLT3CART as a highly effective immunotherapeutic strategy for both FLT3-mutant AML and KMT2A-R ALL that is poised for further investigation and clinical translation.

Survival of infants with KMT2A-rearranged acute lymphoblastic leukemia (ALL) remains dismal despite intensive chemotherapy. We observed constitutive phosphorylation of spleen tyrosine kinase (SYK) and associated signaling proteins in infant ALL patient-derived xenograft (PDX) model specimens and hypothesized that the SYK inhibitor entospletinib would inhibit signaling and cell growth in vitro and leukemia proliferation in vivo. We further predicted that combined entospletinib and chemotherapy could augment anti-leukemia effects. Basal kinase signaling activation and HOXA9/MEIS1 expression differed among KMT2A-rearranged (KMT2A-AFF1 [n=4], KMT2A-MLLT3 [n=1], KMT2A-MLLT1 [n=4]) and non-KMT2A-rearranged [n=3] ALL specimens and stratified by genetic subgroup. Incubation of KMT2A-rearranged ALL cells in vitro with entospletinib inhibited methylcellulose colony formation and SYK pathway signaling in a dose-dependent manner. In vivo inhibition of leukemia proliferation with entospletinib monotherapy was observed in RAS-wild-type KMT2A-AFF1, KMT2A-MLLT3, and KMT2A-MLLT1 ALL PDX models with enhanced activity in combination with vincristine chemotherapy in several models. Surprisingly, entospletinib did not decrease leukemia burden in two KMT2A-AFF1 PDX models with NRAS or KRAS mutations, suggesting potential RAS-mediated resistance to SYK inhibition. As hypothesized, superior inhibition of ALL proliferation was observed in KMT2A-AFF1 PDX models treated with entospletinib and the MEK inhibitor selumetinib versus vehicle or inhibitor monotherapies (p<0.05). In summary, constitutive activation of SYK and associated signaling occurs in KMT2A-rearranged ALL with in vitro and in vivo sensitivity to entospletinib. Combination therapy with vincristine or selumetinib further enhanced treatment effects of SYK inhibition. Clinical study of entospletinib and chemotherapy or other kinase inhibitors in patients with KMT2A-rearranged leukemias may be warranted.

Purpose: Systems biology approaches can identify critical targets in complex cancer signaling networks to inform new therapy combinations that may overcome conventional treatment resistance. Experimental Design: We performed integrated analysis of 1,046 childhood B-ALL cases and developed a data-driven network controllability-based approach to identify synergistic key regulator targets in Philadelphia chromosome-like B-acute lymphoblastic leukemia (Ph-like B-ALL), a common high-risk leukemia subtype associated with hyperactive signal transduction and chemoresistance. Results: We identified 14 dysregulated network nodes in Ph-like ALL involved in aberrant JAK/STAT, Ras/MAPK, and apoptosis pathways and other critical processes. Genetic co-targeting of the synergistic key regulator pair STAT5B and BCL2-associated athanogene 1 (BAG1) significantly reduced leukemia cell viability in vitro. Pharmacologic inhibition with dual small molecule inhibitor therapy targeting this pair of key nodes further demonstrated enhanced anti-leukemia efficacy of combining the BCL-2 inhibitor venetoclax with the tyrosine kinase inhibitors ruxolitinib or dasatinib in vitro in human Ph-like ALL cell lines and in vivo in multiple childhood Ph-like ALL patient-derived xenograft models. Consistent with network controllability theory, co-inhibitor treatment also shifted the transcriptomic state of Ph-like ALL cells to become less like kinase-activated BCR-ABL1-rearranged (Ph+) B-ALL and more similar to prognostically-favorable childhood B-ALL subtypes. Conclusions: Our study represents a powerful conceptual framework for combinatorial drug discovery based on systematic interrogation of synergistic vulnerability pathways with pharmacologic inhibitor validation in preclinical human leukemia models.

PI3K/mTOR signaling is commonly dysregulated in acute lymphoblastic leukemia (ALL). The TACL2014-001 phase 1 trial of the mTOR inhibitor temsirolimus in combination with cyclophosphamide and etoposide was performed in children and adolescents with relapsed/refractory ALL. Temsirolimus was administered intravenously (IV) on days 1 and 8 with cyclophosphamide 440 mg/m2 and etoposide 100 mg/m2 IV daily days 1-5. The starting dose of temsirolimus was 7.5 mg/m2 (DL1) with escalation to 10 mg/m2 (DL2), 15 mg/m2 (DL3), and 25 mg/m2 (DL4). PI3K/mTOR pathway inhibition was measured by phosphoflow cytometry analysis of peripheral blood specimens from treated patients. Sixteen heavily-pretreated patients were enrolled with 15 evaluable for toxicity. One dose-limiting toxicity (DLT) of grade 4 pleural and pericardial effusions occurred in a patient treated at DL3. Additional DLTs were not seen in the DL3 expansion or DL4 cohort. Grade 3/4 non-hematologic toxicities occurring in ≥3 patients included febrile neutropenia, elevated alanine aminotransferase, hypokalemia, mucositis, and tumor lysis syndrome and occurred across all DLs. Complete responses were observed at all DLs with a 47% overall response rate and 27% complete response rate. Pharmacodynamic correlative studies demonstrated dose-dependent inhibition of PI3K/mTOR pathway phosphoproteins in all studied patients. Temsirolimus at doses up to 25 mg/m2 with cyclophosphamide and etoposide had an acceptable safety profile in children with relapsed/refractory ALL. Responses were observed across all DLs. Pharmacodynamic mTOR target inhibition was achieved and appeared to correlate with temsirolimus dose. Future testing of next-generation PI3K/mTOR pathway inhibitors with chemotherapy may be warranted to increase response rates in children with relapsed/refractory ALL.

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