Tyrosine kinases in KMT2A/MLL-rearranged acute leukemias as potential therapeutic targets to overcome cancer drug resistance

Aim: The main goal of this study was to elucidate at the transcript level the tyrosine kinase expression profiles of primary leukemia cells from mixed lineage leukemia 1 gene rearranged (KMT2A/MLL-R+) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients.Methods: We evaluated protein tyrosine kinase (PTK) gene expression profiles of primary leukemic cells in KMT2A/MLL-R+ AML and ALL patients using publicly available archived datasets. Results: Our studies provided unprecedented evidence that the genetic signatures of KMT2A/MLL-R+ AML and ALL cells are characterized by transcript-level overexpression of specific PTK. In infants, children and adults with KMT2A/MLL-R+ ALL, as well as pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family kinase TYK2, and the SRC family kinases FGR and HCK were differentially amplified. Conclusion: These results provide new insights regarding the clinical potential of small molecule inhibitors of these PTK, many of which are already FDA/EMA-approved for other indications, as components of innovative multi-modality treatment platforms against KMT2A/MLL-R+ acute leukemias

Identification and targeting of CD22ΔE12 as a molecular RNAi target to overcome drug resistance in high-risk B-lineage leukemias and lymphomas

Aim: CD22ΔE12 as an oncogenic driver lesion in aggressive and drug-resistant B-precursor acute lymphoblastic leukemia (BPL) cells. The purpose of the present study was to identify the CD22ΔE12-specific signature transcriptome in human BPL cells and evaluate the clinical potential of a nanoscale formulation of CD22ΔE12-siRNA as an RNAi therapeutic against drug-resistant BPL. CD22ΔE12-siRNA nanoparticles significantly improved the event-free survival (EFS) outcome of NOD/SCID (NS) mice challenged with human BPL xenograft cells.Methods: Gene expression and translational bioinformatics methods were applied to examine the expression of the CD22ΔE12-specific signature transcriptome in human BPL cells in subsets of BPL patients. Survival analysis for mice challenged with BPL cells and treated with CD22ΔE12 siRNA was performed using standard methods.Results: Leukemia cells from CD22ΔE12-Tg mice exhibit gene and protein expression profiles consistent with constitutive activation of multiple signaling networks, mimicking the profiles of relapsed BPL patients as well as newly diagnosed high-risk patients with BCR-ABL+/Philadelphia chromosome (Ph)+ BPL as well as Ph-like BPL. A nanoscale formulation of CD22ΔE12-siRNA abrogated the in vivo clonogenicity of the leukemia-initiating leukemic cell fraction in xenograft specimens derived from patients with relapsed BPL and significantly improved the EFS outcome of NS mice challenged with drug-resistant human BPL xenograft cells.Conclusion: The CD22-RNAi technology is applicable to all BPL patients both high risk and standard risk. That is because CD22ΔE12 is a characteristic feature of drug-resistant leukemic clones that escape chemotherapy and cause relapse in both high risk and low risk subgroups of patients. The technology therefore has the potential (1) for prevention of relapses by selectively killing the clones that are most likely to escape chemotherapy and cause relapse as well (2) for treatment of relapses in BPL. This research project may also lead to innovative salvage regimens against other forms of CD22ΔE12-positive relapsed B-lineage leukemias and lymphomas

Wilms’ tumor gene (WT1) is strongly expressed in high-risk subsets of pediatric acute lymphoblastic leukemia

Aim: The purpose of the present study was to perform a comprehensive analysis of WT1 gene expression in high-risk pediatric acute lymphoblastic leukemia (ALL).Methods: We performed a meta-analysis of WT1 gene expression for normal hematopoietic cells vs. primary leukemia cells from 801 pediatric ALL samples deposited in the Oncomine database combined with an in-depth gene expression analysis using our in-house database of gene expression profiles of primary leukemia cells from 1416 pediatric ALL cases. We also examined the expression of WT1 in primary leukemic cells from 299 T-lineage ALL patients in the Oncomine database and 189 T-lineage ALL patients in the archived datasets GSE13159, GSE13351, and GSE13159.Results: Our data provide unprecedented evidence that primary leukemia cells from patients with MLL gene rearrangements (MLL-R) express highest levels of WT1 expression within the high-risk subsets of pediatric B-lineage ALL. Notably, MLL-R+ patients exhibited > 6-fold higher expression levels of the WT1 gene compared to the other B-lineage ALL subtypes combined (P < 0.0001). Our findings in 97 MLL-R+ infant B-lineage ALL cases uniquely demonstrated that WT1 is expressed at 1.5-4.2-fold higher levels in MLL-R+ infant leukemia cells than in normal hematopoietic cells and revealed that WT1 expression level was substantially higher in steroid-resistant infant leukemia cells when compared to non-leukemic healthy bone marrow cells. Furthermore, our study demonstrates for the first time that the WT1-regulated EWSR1, TP53, U2AF2, and WTAP genes (i.e., WT1 interactome) were differentially upregulated in MLL-R+ leukemia cells illustrating that the MLL-regulatory pathway is aberrantly upregulated in MLL-R+ pediatric B-lineage ALL. These novel insights provide a compelling rationale for targeting WT1 in second line treatment of MLL-R+ pediatric B-lineage ALL, including MLL-R+ infant ALL. Furthermore, our study is the first to demonstrate that leukemia cells from 370 Ph-like patients had significantly higher WT1 expression when compared to normal hematopoietic cells. Finally, our findings demonstrate for the first time that chemotherapy-resistant primarily leukemic cells from relapsed B-lineage ALL patients exhibit higher expression levels of WT1 than primary leukemia cells from newly diagnosed B-lineage ALL patients (P = 0.001).Conclusion: Our findings indicate that the WT1 gene product may serve as a target for immunotherapy in high risk/poor prognosis subsets of newly diagnosed as well as relapsed pediatric B-lineage ALL. Our findings also significantly expand the current knowledge of WT1 expression in T-lineage ALL and provide new evidence that WT1 gene and its interactome are expressed in T-lineage ALL cells at significantly higher levels than in normal hematopoietic cells. This previously unknown differential expression profile uniquely indicates that the protein product of WT1 would be an attractive molecular target for treatment of T-lineage ALL as well

Upregulated Expression of ERBB2/HER2 in Multiple Myeloma as a Predictor of Poor Survival Outcomes

The main goal of the present study was to examine if the RNA-sequencing (RNAseq)-based ERBB2/HER2 expression level in malignant plasma cells from multiple myeloma (MM) patients has clinical significance for treatment outcomes and survival. We examined the relationship between the RNAseq-based ERBB2 messenger ribonucleic acid (mRNA) levels in malignant plasma cells and survival outcomes in 787 MM patients treated on contemporary standard regimens. ERBB2 was expressed at significantly higher levels than ERBB1 as well as ERBB3 across all three stages of the disease. Upregulated expression of ERBB2 mRNA in MM cells was correlated with amplified expression of mRNAs for transcription factors (TF) that recognize the ERBB2 gene promoter sites. Patients with higher levels of ERBB2 mRNA in their malignant plasma cells experienced significantly increased cancer mortality, shorter progression-free survival, and worse overall survival than other patients. The adverse impact of high ERBB2 expression on patient survival outcomes remained significant in multivariate Cox proportional hazards models that accounted for the effects of other prognostic factors. To the best of our knowledge, this is the first demonstration of an adverse prognostic impact of high-level ERBB2 expression in MM patients. Our results encourage further evaluation of the prognostic significance of high-level ERBB2 mRNA expression and the clinical potential of ERBB2-targeting therapeutics as personalized medicines to overcome cancer drug resistance in high-risk as well as relapsed/refractory MM

Contemporary patient-tailored treatment strategies against high risk and relapsed or refractory multiple myeloma

Recurrence of disease due to chemotherapy drug resistance remains a major obstacle to a more successful survival outcome of multiple myeloma (MM). Overcoming drug resistance and salvaging patients with relapsed and/or refractory (R/R) MM is an urgent and unmet medical need. Several new personalized treatment strategies have been developed against molecular targets to overcome this drug resistance. There are several targeted therapeutics with anti-MM activity in clinical pipeline, including inhibitors of anti-apoptotic proteins, monoclonal antibodies, antibody-drug conjugates, bispecific antibodies, fusion proteins, and various cell therapy platforms. For example, B-cell maturation antigen (BCMA)-specific CAR-T cell platforms showed promising activity in heavily pretreated R/R MM patients. Therefore, there is renewed hope for high-risk as well as R/R MM patients in the era of personalized medicine. Keywords: Personalized medicine, Multiple myeloma, Stem cell transplantation, Immuno-oncology, Immunotherapy, Biotherap

Liposomal Nanoparticles of a Spleen Tyrosine Kinase P-Site Inhibitor Amplify the Potency of Low Dose Total Body Irradiation Against Aggressive B-Precursor Leukemia and Yield Superior Survival Outcomes in Mice

This study was designed to improve the efficacy of radiation therapy against radiation-resistant leukemia. We report that the potency of low dose radiation therapy against B-precursor acute lymphoblastic leukemia (BPL) can be markedly enhanced by combining radiation with a liposomal nanoparticle (LNP) formulation of the SYK-P-site inhibitor C61 (“C61-LNP”). C61-LNP plus low dose total body irradiation (TBI) was substantially more effective than TBI alone or C61-LNP alone in improving the event-free survival outcome NOD/SCID mice challenged with an otherwise invariably fatal dose of human ALL xenograft cells derived from relapsed BPL patients. C61-LNP plus low dose TBI also yielded progression-free survival, tumor-free survival and overall survival outcomes in CD22ΔE12×BCR–ABL double transgenic mice with advanced stage, radiation-resistant BPL with lymphomatous features that were significantly superior to those of mice treated with TBI alone or C61-LNP alone

A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies

The purposes of the present study were to further evaluate the biologic significance of the CD22ΔE12 molecular lesion and determine if it could serve as a molecular target for RNA interference (RNAi) therapy. We show that both pediatric and adult B-lineage lymphoid malignancies are characterized by a very high incidence of the CD22ΔE12 genetic defect. We provide unprecedented experimental evidence for a previously unrecognized causal link between CD22ΔE12 and aggressive biology of BPL cells by demonstrating that siRNA-mediated knockdown of CD22ΔE12 in primary BPL cells is associated with a marked inhibition of their clonogenicity. These findings provide the preclinical proof-of-concept that siRNA-mediated depletion of CD22ΔE12 may help develop effective treatments for high-risk and relapsed BPL patients who are in urgent need for therapeutic innovations. We also describe a unique polypeptide-based nanoparticle formulation of CD22ΔE12-siRNA as an RNAi therapeutic candidate targeting CD22ΔE12 that is capable of delivering its siRNA cargo into the cytoplasm of leukemia cells causing effective CD22ΔE12 depletion and marked inhibition of leukemic cell growth. Further development and optimization of this nanoparticle or other nanoformulation platforms for CD22ΔE12-siRNA may facilitate the development of an effective therapeutic RNAi strategy against a paradigm shift in therapy of aggressive or chemotherapy-resistant B-lineage lymphoid malignancies

Low Dose Total Body Irradiation Combined With Recombinant CD19-Ligand × Soluble TRAIL Fusion Protein is Highly Effective Against Radiation-resistant B-precursor Acute Lymphoblastic Leukemia in Mice

In high-risk remission B-precursor acute lymphoblastic leukemia (BPL) patients, relapse rates have remained high post-hematopoietic stem cell transplantation (HSCT) even after the use of very intensive total body irradiation (TBI)-based conditioning regimens, especially in patients with a high “minimal residual disease” (MRD) burden. New agents capable of killing radiation-resistant BPL cells and selectively augmenting their radiation sensitivity are therefore urgently needed. We report preclinical proof-of-principle that the potency of radiation therapy against BPL can be augmented by combining radiation with recombinant human CD19-Ligand × soluble TRAIL (“CD19L–sTRAIL”) fusion protein. CD19L–sTRAIL consistently killed radiation-resistant primary leukemia cells from BPL patients as well as BPL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. Low dose total body irradiation (TBI) combined with CD19L–sTRAIL was highly effective against (1) xenografted CD19+ radiochemotherapy-resistant human BPL in NOD/SCID (NS) mice challenged with an otherwise invariably fatal dose of xenograft cells derived from relapsed BPL patients as well as (2) radiation-resistant advanced stage CD19+ murine BPL with lymphomatous features in CD22ΔE12xBCR-ABL double transgenic mice. We hypothesize that the incorporation of CD19L–sTRAIL into the pre-transplant TBI regimens of patients with very high-risk BPL will improve their survival outcome after HSCT