Loss of Ikaros DNA-binding function confers integrin-dependent survival on pre-B cells and progression to acute lymphoblastic leukemia.

Deletion of the DNA-binding domain of the transcription factor Ikaros generates dominant-negative isoforms that interfere with its activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL). Here we found that conditional inactivation of the Ikaros DNA-binding domain in early pre-B cells arrested their differentiation at a stage at which integrin-dependent adhesion to niches augmented signaling via mitogen-activated protein kinases, proliferation and self-renewal and attenuated signaling via the pre-B cell signaling complex (pre-BCR) and the differentiation of pre-B cells. Transplantation of polyclonal Ikaros-mutant pre-B cells resulted in long-latency oligoclonal pre-B-ALL, which demonstrates that loss of Ikaros contributes to multistep B cell leukemogenesis. Our results explain how normal pre-B cells transit from a highly proliferative and stroma-dependent phase to a stroma-independent phase during which differentiation is enabled, and suggest potential therapeutic strategies for Ikaros-mutant B-ALL.

Superenhancer reprogramming drives a B-cell-epithelial transition and high-risk leukemia.

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem-epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem-epithelial-B-cell phenotype that underlies high-risk B-ALL.

Perspective: Pivotal translational hematology and therapeutic insights in chronic myeloid hematopoietic stem cell malignancies.

Despite much of the past 2 years being engulfed by the devastating consequences of the SAR-CoV-2 pandemic, significant progress, even breathtaking, occurred in the field of chronic myeloid malignancies. Some of this was show-cased at the 15th Post-American Society of Hematology (ASH) and the 25th John Goldman workshops on myeloproliferative neoplasms (MPN) held on 9th-10th December 2020 and 7th-10th October 2021, respectively. The inaugural Post-ASH MPN workshop was set out in 2006 by John Goldman (deceased) and Tariq Mughal to answer emerging translational hematology and therapeutics of patients with these malignancies. Rather than present a resume of the discussions, this perspective focuses on some of the pivotal translational hematology and therapeutic insights in these diseases.

Illuminating novel biological aspects and potential new therapeutic approaches for chronic myeloproliferative malignancies.

This review reflects the presentations and discussion at the 14th post-American Society of Hematology (ASH) International Workshop on Chronic Myeloproliferative Malignancies, which took place on the December 10 and 11, 2019, immediately after the 61st ASH Annual Meeting in Orlando, Florida. Rather than present a resume of the proceedings, we address some of the topical translational science research and clinically relevant topics in detail. We consider how recent studies using single-cell genomics and other molecular methods reveal novel aspects of hematopoiesis which in turn raise the possibility of new therapeutic approaches for patients with myeloproliferative neoplasms (MPNs). We discuss how alternative therapies could benefit patients with chronic myeloid leukemia who develop BCR-ABL1 mutant subclones following ABL1-tyrosine kinase inhibitor therapy. In MPNs, we focus on efforts beyond JAK-STAT and the merits of integrating activin receptor ligand traps, interferon-α, and allografting in the current treatment algorithm for patients with myelofibrosis.

Emerging translational science discoveries, clonal approaches, and treatment trends in chronic myeloproliferative neoplasms.

The 60th American Society of Hematology (ASH) held in San Diego in December 2018 was followed by the 13th Post-ASH chronic myeloproliferative neoplasms (MPNs) workshop on December 4 and 5, 2018. This closed annual workshop, first introduced in 2006 by Goldman and Mughal, was organized in collaboration with Alpine Oncology Foundation and allowed experts in preclinical and clinical research in the chronic MPNs to discuss the current scenario, including relevant presentations at ASH, and address pivotal open questions that impact translational research and clinical management. This review is based on the presentations and deliberations at this workshop, and rather than provide a resume of the proceedings, we have selected some of the important translational science and treatment issues that require clarity. We discuss the experimental and observational evidence to support the intimate interaction between aging, inflammation, and clonal evolution of MPNs, the clinical impact of the unfolding mutational landscape on the emerging targets and treatment of MPNs, new methods to detect clonal heterogeneity, the challenges in managing childhood and adolescent MPN, and reflect on the treatment of systemic mastocytosis (SM) following the licensing of midostaurin.

Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1.

Tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph(+)B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GRB2-associated binding protein 2 (GAB2) as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1-evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1-transducedGab2(-/-)bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors phosphatidylinositol 3-kinase (PI3K) or SRC homology 2-containing phosphotyrosine phosphatase 2 (SHP2). GAB2 deficiency also attenuated BCR-ABL1-induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2-SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph(+)hematologic neoplasms.

Precision immunotherapy, mutational landscape, and emerging tools to optimize clinical outcomes in patients with classical myeloproliferative neoplasms.

Following the 47th American Society of Hematology Meeting in 2005, the late John Goldman and Tariq Mughal commenced a conference, the 1st Post-ASH Workshop, which brought together clinicians and scientists, to accelerate the adoption of new therapies for patients with myeloproliferative neoplasms (MPNs). The concept began with recognition of the CML success story following imatinib therapy, the discovery of JAK2V617F , and the demonstration that BCR-ABL1-negative MPNs are driven by abnormal JAK2 activation. This review is based on the presentations and deliberations at the XIIth Post-ASH Workshop on BCR-ABL1 positive and negative MPNs that took place on December 12 to 13, 2017, in Atlanta, Georgia, immediately following the 59th American Society of Hematology Meeting. We have selected some of the translational research and clinical topics, rather than an account of the proceedings. We discuss the role of immunotherapy in MPNs and the impact of the mutational landscape on TKI treatment in CML. We also consider how we might reduce TKI cardiovascular side effects, the potential role of nutrition as adjunctive nonpharmacologic intervention to reduce chronic inflammation in MPNs, and novel investigational therapies for MPNs.

Phase 1 dose-finding study of rebastinib (DCC-2036) in patients with relapsed chronic myeloid leukemia and acute myeloid leukemia.

A vailable tyrosine kinase inhibitors for chronic myeloid leukemia bind in an adenosine 5'-triphosphate-binding pocket and are affected by evolving mutations that confer resistance. Rebastinib was identified as a switch control inhibitor of BCR-ABL1 and FLT3 and may be active against resistant mutations. A Phase 1, first-in-human, single-agent study investigated rebastinib in relapsed or refractory chronic or acute myeloid leukemia. The primary objectives were to investigate the safety of rebastinib and establish the maximum tolerated dose and recommended Phase 2 dose. Fifty-seven patients received treatment with rebastinib. Sixteen patients were treated using powder-in-capsule preparations at doses from 57 mg to 1200 mg daily, and 41 received tablet preparations at doses of 100 mg to 400 mg daily. Dose-limiting toxicities were dysarthria, muscle weakness, and peripheral neuropathy. The maximum tolerated dose was 150 mg tablets administered twice daily. Rebastinib was rapidly absorbed. Bioavailability was 3- to 4-fold greater with formulated tablets compared to unformulated capsules. Eight complete hematologic responses were achieved in 40 evaluable chronic myeloid leukemia patients, 4 of which had a T315I mutation. None of the 5 patients with acute myeloid leukemia responded. Pharmacodynamic analysis showed inhibition of phosphorylation of substrates of BCR-ABL1 or FLT3 by rebastinib. Although clinical activity was observed, clinical benefit was insufficient to justify continued development in chronic or acute myeloid leukemia. Pharmacodynamic analyses suggest that other kinases inhibited by rebastinib, such as TIE2, may be more relevant targets for the clinical development of rebastinib (clinicaltrials.gov Identifier:00827138).

IKK-dependent activation of NF-κB contributes to myeloid and lymphoid leukemogenesis by BCR-ABL1.

The product of the Ph chromosome, the BCR-ABL1 tyrosine kinase activates diverse signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph(+) B-cell acute lymphoblastic leukemia (B-ALL). Previous studies showed that nuclear factor κB (NF-κB) is activated in BCR-ABL1-expressing cells, but the mechanism of activation and importance of NF-κB to the pathogenesis of BCR-ABL1-positive myeloid and lymphoid leukemias are unknown. Coexpression of BCR-ABL1 and a superrepressor mutant of inhibitory NF-κB α (IκBαSR) blocked nuclear p65/RelA expression and inhibited the proliferation of Ba/F3 cells and primary BCR-ABL1-transformed B lymphoblasts without affecting cell survival. In retroviral mouse models of CML and B-ALL, coexpression of IκBαSR attenuated leukemogenesis, prolonged survival, and reduced myeloid leukemic stem cells. Coexpression of dominant-negative mutants of IκB kinase α (IKKα)/IKK1 or IKKß/IKK2 also inhibited lymphoid and myeloid leukemogenesis by BCR-ABL1. Blockade of NF-κB decreased expression of the NF-κB targets c-MYC and BCL-X and increased the sensitivity of BCR-ABL1-transformed lymphoblasts to ABL1 kinase inhibitors. These results demonstrate that NF-κB is activated through the canonical IKK pathway and plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL1, validating NF-κB and IKKs as targets for therapy of Ph(+) leukemias.

Selectins and their ligands are required for homing and engraftment of BCR-ABL1+ leukemic stem cells in the bone marrow niche.

We investigated adhesion pathways that contribute to engraftment of breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1)-induced chronic myelogenous leukemia (CML)-like myeloproliferative neoplasia in a mouse retroviral transduction/transplantation model. Compared with normal stem/progenitor cells, BCR-ABL1(+) progenitors had similar expression of very late antigen-4 (VLA4), VLA5, leukocyte functional antigen-1, and CXCR4 but lower expression of P-selectin glycoprotein ligand-1 (PSGL-1) and of L-selectin. Whereas vascular cell adhesion molecule-1 and P-selectin were not required, deficiency of E-selectin in the recipient bone marrow endothelium significantly reduced engraftment by BCR-ABL1-expressing stem cells following intravenous injection, with leukemogenesis restored by direct intrafemoral injection. BCR-ABL1-expressing cells deficient for PSGL-1 or the selectin ligand-synthesizing enzymes core-2 ß1,6-N-acetylglucosaminyltransferase or fucosyltransferases IV/VII were impaired for engraftment, and destruction of selectin ligands on leukemic progenitors by neuraminidase reduced engraftment. BCR-ABL1-expressing L-selectin-deficient progenitors were also defective in homing and engraftment, with leukemogenesis rescued by coexpression of chimeric E/L-selectin. Antibody to L-selectin decreased the engraftment of BCR-ABL1-transduced stem cells. These results establish that BCR-ABL1(+) leukemic stem cells rely to a greater extent on selectins and their ligands for homing and engraftment than do normal stem cells. Selectin blockade is a novel strategy to exploit differences between normal and leukemic stem cells that may be beneficial in autologous transplantation for CML and perhaps other leukemias.

Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition.

Cancer therapy has progressed remarkably in recent years. In no area has this been more apparent than in the development of "targeted therapies", particularly those using drugs that inhibit the activity of certain tyrosine kinases, activating mutations or amplifications of which are causal, or strongly contributory, to tumorigenesis. However, some of these therapies have been associated with toxicity to the heart. Here we summarize what is known about the cardiotoxicity of cancer drugs that target tyrosine kinases. We focus on basic mechanisms through which interruption of specific signalling pathways leads to cardiomyocyte dysfunction and/or death, and contrast this with therapeutic responses in cancer cells.

Distinct graft-versus-leukemic stem cell effects of early or delayed donor leukocyte infusions in a mouse chronic myeloid leukemia model.

Among hematologic neoplasms, chronic myeloid leukemia (CML) is exquisitely sensitive to graft-versus-leukemia (GVL) because patients relapsing after allogeneic hematopoietic stem-cell transplantation (alloHSCT) can be cured by donor leukocyte infusion (DLI); however, the cellular mechanisms and strategies to separate GVL from GVHD are unclear. We used a BCR-ABL1 transduction/transplantation mouse model to study the mechanisms of DLI in MHC-matched, minor histocompatibility antigen-mismatched allogeneic chimeras with CML-like leukemia, in which DLI can be administered at the time of transplantation (early) or after recovery of hematopoiesis (delayed). After early DLI, CML-like leukemia cannot be transferred into immunocompetent secondary recipients as soon as 4 days after primary transplantation, demonstrating that cotransplantation of T lymphocytes blocks the engraftment of BCR-ABL1-transduced stem cells. In contrast, in allogeneic chimeras with established CML-like leukemia, combined treatment with delayed DLI and the kinase inhibitor imatinib eradicates leukemia with minimal GVHD. The GVL effect is directed against minor histocompatibility antigens shared by normal and leukemic stem cells, and is mediated predominantly by CD8+ T cells, with minor contributions from CD5- splenocytes, including natural killer cells. These results define a physiologic model of adoptive immunotherapy of CML that will be useful for investigating the cellular and molecular mechanisms of GVL.

Essential role for Stat5a/b in myeloproliferative neoplasms induced by BCR-ABL1 and JAK2(V617F) in mice.

STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the Stat5a/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2(V617F) in retroviral transplantation models of CML and PV. Loss of one Stat5a/b allele resulted in a decrease in BCR-ABL1-induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of Stat5a/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2(V617F) failed to induce polycythemia in recipients after deletion of Stat5a/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5a/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2(V617F), and validate STAT5a/b and the genes they regulate as targets for therapy in these MPNs.

Targeting CXCR4 with cell-penetrating pepducins in lymphoma and lymphocytic leukemia.

The chemokine receptor CXCR4, which normally regulates stromal stem cell interactions in the bone marrow, is highly expressed on a variety of malignant hematologic cells, including lymphoma and lymphocytic leukemias. A new treatment concept has arisen wherein CXCR4 may be an effective therapeutic target as an adjunct to treatment of hematologic neoplasms with chemo- and immunotherapy. In the present study, we developed pepducins, cell-penetrating lipopeptide antagonists of CXCR4, to interdict CXCL12-CXCR4 transmembrane signaling to intracellular G-proteins. We demonstrate that pepducins targeting the first (i1) or third (i3) intracellular loops of CXCR4 completely abrogate CXCL12-mediated cell migration of lymphocytic leukemias and lymphomas. Stromal-cell coculture protects lymphoma cells from apoptosis in response to treatment with the CD20-targeted Ab rituximab. However, combination treatment with CXCR4 pepducins and rituximab significantly increases the apoptotic effect of rituximab. Furthermore, treatment of mice bearing disseminated lymphoma xenografts with pepducins alone or in combination with rituximab significantly increased their survival. These data demonstrate that CXCL12-CXCR4 signaling can be effectively inhibited by cell-penetrating pepducins, which represents a potential new treatment strategy for lymphoid malignancies.

Oncogenic signaling: new insights and controversies from chronic myeloid leukemia.

Chronic myeloid leukemia (CML), which is caused by the BCR-ABL fusion tyrosine kinase, is one of the most intensively studied human cancers. ABL kinase inhibitors have been spectacularly successful in treating CML, but disease persistence and acquired drug resistance can prevent eradication and cure of the leukemia. The development of better therapies will depend on a full understanding of signaling pathways in CML, facilitated by model studies using mutant mice.

CDK6 kinase activity is required for thymocyte development.

Cyclin-dependent kinase-6 (CDK6) is required for early thymocyte development and tumorigenesis. To mechanistically dissect the role of CDK6 in thymocyte development, we generated and analyzed mutant knock-in mice and found that mice expressing a kinase-dead Cdk6 allele (Cdk6(K43M)) had a pronounced reduction in thymocytes and hematopoietic stem cells and progenitor cells (Lin⁻Sca-1⁺c-Kit⁺ [LSK]). In contrast, mice expressing the INK4-insensitive, hyperactive Cdk6(R31C) allele displayed excess proliferation in LSK and thymocytes. However, this is countered at least in part by increased apoptosis, which may limit progenitor and thymocyte expansion in the absence of other genetic events. Our mechanistic studies demonstrate that CDK6 kinase activity contributes to Notch signaling because inactive CDK6 kinase disrupts Notch-dependent survival, proliferation, and differentiation of LSK, with concomitant alteration of Notch target gene expression, such as massive up-regulation of CD25. Further, knockout of CD25 in Cdk6(K43M) mice rescued most defects observed in young mice. These results illustrate an important role for CDK6 kinase activity in thymocyte development that operates partially through modulating Notch target gene expression. This role of CDK6 as a downstream mediator of Notch identifies CDK6 kinase activity as a potential therapeutic target in human lymphoid malignancies.

Autologous stem cell transplant recipients tolerate haploidentical related-donor natural killer cell-enriched infusions.

BACKGROUND: In the setting of allogeneic stem cell transplantation (SCT), infusing natural killer (NK) cells from a major histocompatibility complex (MHC)-mismatched donor can mediate an antileukemic effect. The graft-versus-tumor effect after autologous stem cell transplantation (ASCT) may result in less disease relapse. STUDY DESIGN AND METHODS: We performed a Phase I clinical trial to assess the safety and feasibility of infusing distantly processed donor NK-enriched mononuclear cell (NK-MNC) infusions from a MHC haplotype-mismatched (haploidentical) donor to patients who recently underwent ASCT for a hematologic malignancy. On Day 1, peripheral blood MNCs were obtained by steady-state leukapheresis and sent from Boston to the Production Assistance for Cellular Therapies (PACT) facility at the University of Minnesota, where immunomagnetic depletion of CD3 cells was performed on Day 2. NK-MNC products were then returned to Boston on Day 2 for infusion on Day 3. Toxicity, cellular product characteristics, and logistic events were monitored. RESULTS: At a median of 90 days (range, 49-191 days) after ASCT, 13 patients were treated with escalating doses of NK-MNCs per kilogram from 10(5) to 2 × 10(7) . Adverse effects included Grade 2 rigors and muscle aches, but no Grade 3 or 4 events and no graft-versus-host disease or marrow suppression. One air courier delay occurred. NK-MNC products were viable with cytotoxic activity after transport. CONCLUSION: CD3-depleted, MHC-mismatched allogeneic NK-MNC infusions can be safely and feasibly administered to patients after ASCT after distant processing and transport, justifying further development of this approach.

Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells.

In individuals with chronic myeloid leukemia (CML) treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft contribute to leukemic relapse, but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Here we show that CD44 expression is increased on mouse stem-progenitor cells expressing BCR-ABL and that CD44 contributes functional E-selectin ligands. In a mouse retroviral transplantation model of CML, BCR-ABL1-transduced progenitors from CD44-mutant donors are defective in homing to recipient marrow, resulting in decreased engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engraft as efficiently as do wild-type HSCs. CD44 is dispensable for induction of acute B-lymphoblastic leukemia by BCR-ABL, indicating that CD44 is specifically required on leukemic cells that initiate CML. The requirement for donor CD44 is bypassed by direct intrafemoral injection of BCR-ABL1-transduced CD44-deficient stem cells or by coexpression of human CD44. Antibody to CD44 attenuates induction of CML-like leukemia in recipients. These results show that BCR-ABL-expressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML.

Cardiotoxicity of the cancer therapeutic agent imatinib mesylate.

Imatinib mesylate (Gleevec) is a small-molecule inhibitor of the fusion protein Bcr-Abl, the causal agent in chronic myelogenous leukemia. Here we report ten individuals who developed severe congestive heart failure while on imatinib and we show that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib show mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib.